QR 

41 
B4- 


UC-NRI 


BIOLOGY 

LIBRARY 

G 


publications 


OF  THE 


"University  of /Pennsylvania. 


1Rew  Series,  INo.  4. 


Contributions 

from 

The  Laboratory  of  Hygiene. 

No.   1-2. 

The   Biological  Relation  between    Bacteria   and   the    More 
Highly  Organized  Flora  of   Running  Streams. 

D.   H.   BERGEY,  M.  D. 

Comparative   Studies   upon    the    Pseudo-diphtheria,    or 

Hofmann  Bacillus,   the  Xerosis    Bacillus,   and  the 

Loffler  Bacillus. 

D.   H.   BERGEY.  M.  D. 


PUBLISHED  FOR  THE  UNIVERSITY  OF  PENNSYLVANIA. 
PHILADELPHIA, 

1898. 


List  of  Publications  at  the  close  of 
this  number. 


Orders  for  single  numbers  or  complete  sets 
of  the  Publications  should  be  sent  direct  to 
J.  HARTLEY  MERRICK,  Assistant  Secretary, 
College  Hall,  University  of  Pennsylvania,  Sta- 
tion B,  Philadelphia,  Pa. 


( 
{publications 

OF  THE 

"(University  of  Pennsylvania, 

Hew  Serfea,  Ho.  4* 


Contributions 

from 

The  Laboratory  of  Hygiene. 

No.  1-2. 

The  Biological  Relation  between   Bacteria  and  the   More 
Highly  Organized  Flora  of  Running  Streams. 

D.  H.  BERGEY,  M.  D. 

Comparative   Studies   upon   the   Pseudo-diphtheria,    or 

Hofmann  Bacillus,  the  Xerosis   Bacillus,  and  the 

Loffler  Bacillus. 

D.  H.  BERGEY,  M.  D. 


PUBLISHED  FOR  THE  UNIVERSITY  OF  PENNSYLVANIA. 

PHILADELPHIA. 

1898. 


BIOLOGY 

UBRARY 

G 


THE 
BIOLOGICAL  RELATION  BETWEEN  BACTERIA 

AND  THE 

MORE  HIGHLY  ORGANIZED  FLORA  OF 
RUNNING  STREAMS 


BY 


D.  H.  BERGEY,  M.  D.,  First  Assistant 

Laboratory  of  Hygiene,  University  of  Pennsylvania 


THE  BIOLOGICAL  RELATION  BETWEEN   BACTERIA  AND  THE  MORE 
HIGHLY  ORGANIZED  FLORA  OF  RUNNING  STREAMS. 

By  D.    H.    Bergey,    M.  D. 
First  Assistant,  laboratory  of  Hygiene,  University  of  Pennsylvania. 

[Read  before  the  Biological  Club,  March  7,  1898.] 

All  cryptogamic  plants  may  be  divided  into  two  great 
classes  with  reference  to  the  presence  or  absence  of  chloro- 
phyl  within  the  plant  cells.  When  we  come  to  study  the 
life  history  of  these  two  groups  of  organisms  we  find  that 
their  biological  functions  are  closely  related  to  the  fact 
whether  they  contain  chlorophyl  or  not. 

In  many  respects  the  biological  functions  of  the  two 
groups  of  organisms  are  entirely  different  from  each  other. 
Those  cryptogamic  plants  which  contain  chlorophyl  in 
their  cells  possess  biological  functions  not  markedly  dis- 
similar in  character  to  those  of  the  phanerogamic  plants. 
Those  of  the  cryptogamic  plants  which  are  without  chlo- 
rophyl have  biological  functions  which  are  entirely  differ- 
ent from  those  of  the  phanerogamic  plants. 

As  the  basis  of  our  study  of  the  relation  between  the 
biological  functions  of  bacteria  and  the  more  highly  organ- 
ized flora  of  running  streams  we  shall  take  into  considera- 
tion the  nature  of  their  food  supply.  The  food  supply  of 
the  more  highly  organized  flora  does  not  differ  in  any 
essential  respect  from  that  of  the  phanerogamic  plants. 
They  subsist  on  the  various  mineral  matters  contained  in 
the  soil  and  water  and  upon  the  constituents  of  the  atmos- 
phere. For  the  healthy  action  of  all  their  body  cells  they 
require,  in  addition,  the  presence  of  sunlight.  Their  chlo- 
rophyl-bearing  cells  are  dependent  upon  the  influence  of 
sunlight  for  the  power  of  building  up  the  most  complex 
organic  compounds  and  elaborating  them  into  the  various 

(5) 


6          Biological  Relation  Between  Bacteria  and  the  More 

tissues  and  special  organs.  Their  food  supply  consists  of 
dissolved  or  soluble  salts  of  various  kinds  in  the  soil  and 
water,  and  of  the  carbonic  acid  of  the  atmosphere. 

On  the  other  hand,  the  bacteria  and  other  chlorophyl- 
free  plants  subsist  mainly  upon  nitrogenous  organic  mat- 
ter or  upon  inorganic  compounds  containing  nitrogen, 
such  as  the  various  salts  of  ammonia  and  the  nitrates,  from 
which  they  derive  their  supply  of  nitrogen.  In  most  in- 
stances the  nitrogen  is  most  readily  taken  from  diffusible 
albuminoid  matter;  less  easily  from  the  ammonia  com- 
binations. 

Nageli  was  able  to  demonstrate  that  a  certain  class  of 
bacteria  known  as  the  denitrifying  bacteria  were  able  to 
bring  about  the  reduction  of  nitrates  and  convert  them 
into  nitrites,  ammonia,  or  even  nitrogen. 

A  quite  exceptional  mode  of  meeting  the  demand  for 
nitrogen  is  found  in  those  bacteria  forming  the  tubercles 
on  the  leguminoseae  and  allied  plants  in  which  they,  in 
symbiosis  with  the  host  plants  on  which  they  live,  are 
capable  of  fixing  the  free  nitrogen  of  the  atmosphere  and 
assimilating  the  same  in  building  up  their  cell  bodies. 

The  supply  of  carbon  of  the  chlorophyl-free  plants,  in 
contradistinction  to  that  of  the  higher  plants,  is  obtained 
by  breaking  up  different  forms  of  carbo-hydrates.  Aside 
from  albumen  and  peptone,  they  use  sugar  and  similar 
carbo-hydrates  and  glycerine,  as  a  source  of  carbon.  They 
are  also  capable  of  using  organic  matters  of  different  chem- 
ical constitution,  as  the  basic  and  diabasic  acids  (vinegar), 
the  hydroxilized  acids  (tartaric  and  citric),  as  asparagin, 
lucin,  the  different  alcohols,  amine,  ester,  urea,  etc.  In 
very  dilute  solutions  they  are  even  capable  of  using  as 
nourishment  compounds  containing  carbon  which,  in  con- 
centrated solutions,  are  distinctly  poisonous,  such  as  car- 
bolic acid  and  salicylic  acid. 

Some  of  the  chlorophyl-bearing  organisms,  such  as  the 
algae,  which  closely  resemble  the  bacteria  in  some  respects, 


Highly  Organized  Flora  of  Running  Streams.  7 

ordinarily  satisfy  their  requirements  for  carbon  and  nitrogen 
by  taking  these  elements  from  CO2  and  NH3,  or  even  HNO8, 
out  of  substances  in  their  environment,  and  converting 
them  into  complex  carbonaceous  and  nitrogenous  matter 
with  the  aid  of  the  chlorophyl.  It  is  possible  for  these 
plants  to  take  their  nourishment  out  of  water  containing 
the  necessary  mineral  substances,  and  out  of  air  containing 
CO2  and  NH3  and  assimilate  the  carbon  and  nitrogen  de- 
rived from  these  sources.  They  also  possess,  to  a  limited 
extent,  the  power  of  breaking  up  organic  matter  and 
derive  their  C  and  N  directly  from  such  matter.  The 
bacteria,  however,  through  the  absence  of  chlorophyl,  are 
incapable  of  existing  in  this  manner,  but  require  previously- 
prepared  organic  substances  to  meet  the  needs  of  their 
bodies,  and  to  form  new  substance.  Therefore,  they  cannot 
exist  in  pure  water  containing  only  mineral  matter.  They 
vegetate  on  dead  organic  matter,  rich  in  carbon  and  nitro- 
gen, and  likewise  on  dead  plants  and  animal  organisms,  or 
they  live  as  parasites  drawing  the  organic  matter  necessary 
for  their  life  and  growth  from  their  vegetable  and  animal 
hosts. 

From  this  one  may  form  an  idea  as  to  the  significance 
of  the  bacteria  in  nature.  In  order  to  provide  continu- 
ously the  simple  nourishment  necessary  for  the  chlorophyl- 
bearing  vegetation,  it  is  essential  to  have  a  constant  reduc- 
tion and  solution  of  this  form  of  vegetable  substances  back 
into  simple  chemical  combinations.  The  entire  yearly 
vegetation  which  has  formed  and  died  must  be  so  changed 
in  a  relatively  short  time  that  the  complicated  vegetable 
substances,  the  albumen,  carbo-hydrates  and  cellulose  are 
again  converted  into  water,  CO2  and  NHS.  Only  under 
such  conditions  is  the  continuous  regeneration  of  the 
higher  vegetation  possible. 

A  part  of  this  work  of  reconstruction  falls  upon  animal 
organisms,  the  animal  cells  breaking  up  the  vegetable 
matter  taken  up,  and  delivering  it  over  for  oxidation. 


8          Biological  Relation  Between  Bacteria  and  the  More 

The  energy  which  is  stored  in  the  complex  chemical  com- 
binations of  the  plants  of  higher  organization  with  the 
aid  of  their  chlorophyl  and  sunlight  is  used  by  the  animal 
organisms  for  the  production  of  animal  heat  and  the 
different  functions  of  the  body. 

With  this  very  brief  consideration  of  the  general  biologi- 
cal functions  of  the  bacteria  and  the  more  highly  organized 
flora,  we  are  in  a  position  to  consider  briefly  the  relation 
which  these  organisms  bear  to  each  other  in  running 
streams.  This  relation  has  been  studied  to  some  extent 
by  experimenters  with  the  view  of  discovering  something 
about  the  modus  operandi  of  the  self-purification  of  pol- 
luted streams.  It  has  been  found  that  the  chlorophyl- 
bearing  organisms  are  confined  largely  to  fresh,  running 
water.  These  organisms  do  not  multiply  very  rapidly, 
and  may  even  die  off,  in  polluted  waters.  The  conditions 
for  growth  in  waters  of  this  character  are  not  very  favor- 
able for  these  organisms.  The  greater  the  degree  of  pol- 
lution the  more  the  action  of  sunlight — the  most  essential 
feature  of  the  life  and  growth  of  chlorophyl-bearing  plants 
— is  interfered  with  in  these  waters.  The  character  of  the 
food  material  is  not  adapted  to  their  growth  because  of  the 
predominance  of  organic  substances  in  the  water.  Besides 
this,  because  of  the  presence  of  large  quantities  of  organic 
matter,  we  have  immense  numbers  and  different  kinds  of 
chlorophyl-free  organisms  in  water  of  this  character,  and 
these  are  doubtless  in  a  certain  degree  antagonistic  to  the 
chlorophyl-bearing  organisms :  so  that  their  growth  is 
interfered  with  in  this  manner. 

As  has  been  said,  the  large  quantity  of  organic  matter 
in  polluted  water,  the  hindrance  of  the  action  of  sunlight, 
and  the  presence  of  suspended  matters,  makes  this  a  favor- 
able soil  for  the  growth  and  multiplication  of  all  chlorophyl- 
free  organisms.  They  find  here  an  abundant  food  supply 
and  abundance  of  oxygen,  and  all  other  conditions  favor- 
able for  growth.  In  the  studies  which  have  been  made 


Highly  Organized  Flora  of  Running  Streams.  9 

on  the  question  of  the  self-purification  of  streams  exami- 
nations of  polluted  waters  have  shown  that  the  organisms 
which  predominate  in  these  waters  are  the  chlorophyl-free 
organisms.  Experiments  have  shown  that  these  are  cap- 
able of  existing  upon  organic  matters  such  as  found  in 
these  polluted  streams.  Certain  forms  have  been  found 
more  constantly  than  others ;  appearing  to  possess  a  pre- 
dilection for  such  water.  For  instance,  Pfeiffer  and  Eisen- 
lohr  (Archiv  fur  Hygiene,  Bd.  14,  p.  90,  1892)  in  their 
investigation  of  the  river  Isar  found  large  quantities  of  a 
particular  form  of  beggiatoa.  Samples  of  water  collected 
at  different  points  of  the  river  were  found  to  contain 
enormous  quantities  of  active  growths  of  beggiatoa  roseo- 
persicina  (Zopf).  Between  the  threads  of  this  organism, 
however,  other  fission  fungi  were  found  in  enormous  num- 
bers, as  yeast,  diatoms,  protozoa — in  fact,  all  possible  forms 
of  lower  vegetable  and  animal  life — forms,  however,  which 
were  not  examined  further  because  they  had  no  signifi- 
cance with  regard  to  the  question  under  consideration. 
From  their  investigations  it  seemed  evident  that  this 
particular  variety  of  beggiatoa  was  present  only  in  polluted 
waters,  and  did  not  penetrate  into  the  fresh  water  streams 
entering  the  river ;  its  great  prevalence  at  any  point 
appeared  to  indicate  the  presence  of  undue  quantities  of 
organic  matter. 

Schenk  (Centralblatt  fur  allgemeine  Gesundheitspflege, 
12.  Jahr.,  p.  365,  1892)  in  his  investigation  on  the  signifi- 
cance of  the  Rhine  vegetation  in  the  self-purification  of 
the  river,  reports  on  several  forms  of  algae  found  adhering 
to  the  stones  along  the  banks  in  large  quantities,  which 
seemed  to  him  to  have  some  influence  upon  the  purification 
of  the  river.  Among  the  chlorophyl-free  plants  he  says 
that  the  saprophytic  water  plants  of  the  first  and  greatest 
importance  are  the  bacteria.  The  two  forms  which  he 
says  require  special  consideration,  from  the  large  numbers 
found,  are  the  beggiatoa  alba  and  the  cladothrix  dicho- 


io        Biological  Relation  Between  Bacteria  and  the  More 

toma.  He  says  that  the  beggiatoa  is  of  great  importance 
with  respect  to  the  subject  under  consideration,  occurring 
in  especially  large  quantities  beneath  the  different  sewer 
openings,  forming  a  slimy  layer,  sometimes  floating  in  the 
water,  and  covering  everything  along  the  banks.  The 
masses  consist  of  innumerable  single  threads  built  up  of 
short  members  which  contain  numerous  sulphur  granules 
in  the  interior  of  the  cells.  Though  distributed  every- 
where the  organism  attained  its  greatest  development  only 
in  sewers  or  in  grossly  polluted  water.  The  cladothrix 
dichotoma  was  usually  found  along  the  banks  in  connec- 
tion with  the  beggiatoa  and  in  connection  with  the  cla- 
dothrix glornerata,  in  polluted  water.  These  fission  fungi 
form  the  most  important  part  of  the  chlorophyl-free  water 
fungi  along  the  polluted  banks. 

With  regard  to  the  algae  he  cannot  coincide  with  the 
opinion  of  M.  von  Pettenkofer,  that  they  play  an  important 
r61e  in  the  purification  of  the  Rhine.  He  states  that  J. 
Ufnemann  had  also  reached  the  conviction  that  the  role  of 
the  algae  in  the  purification  of  rivers  should  not  be  over- 
estimated, because  it  was  established  that  the  green-thread 
algae  and  most  of  the  diatoms  can  exist  only  in  fresh  or 
slightly  polluted  water,  and  that  their  action  would  fail 
where  it  is  most  desired.  It  is  otherwise,  however,  with 
the  chlorophyl-free  and  saprophytic  bacteria.  The  water 
bacteria  in  their  totality  play  the  principle  rdle  in  the 
purification  of  rivers,  as  far  as  living  organisms  are  con- 
cerned. Among  these  the  beggiatoa  alba  have  especial 
significance. 

In  a  brief  report  on  the  self-purification  of  streams,  in 
the  Report  of  the  Massachusetts  State  Board  of  Health  for 
1890,  p.  793,  it  is  stated  that  the  micro-organisms  which 
were  instrumental  in  bringing  about  a  ten-fold  reduction 
of  nitrates  in  the  water  and  great  changes  in  nitrogen 
compounds,  with  almost  total  removal  of  free  ammonia, 
were  "  enormous  growth  of  anabaena,  synedra  and  zoo- 


Highly  Organized  Flora  of  Running  Streams.  1 1 

spores,  together  with  many  other  organisms  in  smaller 
numbers.  The  change  produced  in  the  water  by  these 
organisms  made  the  water  turbid  and  green,  and  conse- 
quently unfit  for  drinking,  on  account  of  the  taste,  odor  and 
appearance.  The  nitrate  compounds  are  rapidly  trans- 
formed by  the  organic  growths,  and  it  seems  reasonable 
to  conclude  that  this  means  the  destruction  of  substances 
undergoing  decomposition  contained  in  the  water."  They 
state  further  that  "  from  a  health  standpoint  this  may 
be  classed  as  an  instance  of  the  self-purification  of 
streams,  even  though  the  water  is  temporarily  rendered 
obnoxious  by  the  organisms  which  have  effected  the 
change." 

Recently  E.  Goldschmidt,  W.  Prausnitz  and  others  have 
published  (Hygienische  Rundschau,  8.  Jahrg.,  p.  161)  an 
important  investigation  on  the  death  of  micro-organisms 
in  the  self-purification  of  rivers.  Their  experiments  were 
made  upon  the  river  Isar,  making  a  continuous  study  of 
the  condition  of  the  Isar  from  1889  to  the  present  time,  by 
themselves  and  other  investigators.  Samples  of  water 
were  collected  at  various  points  of  the  Isar  below  Munich: 
for  instance,  at  upper  or  lower  Fohriug,  at  Ismaning,  at 
Freising,  and  at  Landshut.  In  all  these  investigations  it 
was  found  that  there  is  a  uniform  diminution  in  those 
bacteria  which  grow  on  ordinary  gelatine  media,  in  regular 
gradation  as  one  proceeds  down  the  stream.  The  following 
results  show  fairly  well  the  rate  of  diminution  at  the  dif- 
ferent points,  and  are  taken  from  examinations  made  in 
December,  1893,  showing  at  Fohring  24,097  organisms 
per  cubic  centimeter;  at  Ismaning,  15,065;  at  Freising, 
7134,  and  at  Landshut,  1976. 

As  the  result  of  their  investigation  they  have  reached 
the  following  conclusions : 

i.  The  self-purification  of  rivers,  as  shown  in  the  dis- 
appearance of  impurities,  is  not  influenced  by  the  activity 
of  micro-organisms. 


1 2         Biological  Relation  Between  Bacteria  and  the  More 

2.  The   disappearance   of    the   micro-organisms   which 
grow  on  gelatine  media  in  polluted  streams  takes  place 
during  the  day  as  well  as  night,  and  is  not  influenced  by 
light,  though  sunlight  seems  to  assist  in  killing  off  the 
bacteria.  . . . : , 

3.  The  death  of  micro-organisms  follows  rapidly,  and 
during  the  course  of  twenty  kilometers,  requiring  about 
eight  hours,  50  per  cent  of  the  germs  disappeared. 

Undoubtedly,  the  facts  as  presented  by  this  thorough 
and  practical  investigation  can  be  interpreted  in  several 
different  ways.  In  the  first  place,  it  must  be  borne  in 
mind  that  the  nitrifying  bacteria,  those  which  are  con- 
cerned most  particularly  in  the  purification  of  polluted 
waters,  do  not  grow  on  the  ordinary  gelatine  media :  con- 
sequently these  did  not  enter  into  the  consideration  of  the 
bacteriological  analyses  made  by  these  investigators.  In 
the  second  place,  through  the  action  of  sedimentation,  some 
of  the  organic  matter  subsided  and  took  its  place  on  the 
bottom  of  the  river;  with  the  subsidence  of  the  food 
material  for  the  micro-organisms  it  is  most  evident  that 
they  also  took  the  same  course ;  so  that  in  this  manner  a 
considerable  proportion  of  the  micro-organisms  may  have 
been  removed.  Furthermore,  it  is  altogether  probable 
that  there  was,  and  always  is,  a  marked  reduction  of  those 
bacteria  which  are  capable  of  growing  on  gelatine  media, 
and  that  they  are  destroyed  through  the  purifying  agencies 
at  work  in  flowing  streams. 

The  putrefactive  decomposition  of 'albuminous  material 
of  animal  and  vegetable  origin  is  effected  by  a  great 
variety  of  micro-organisms  and  gives  rise  to  the  formation 
of  a  great  variety  of  products,  some  of  which  are  volatile 
and  characterized  by  their  offensive  odors.  According  to 
Fliigge,  the  first  change  which  occurs  consists  in  the  trans- 
formation of  the  albumens  into  peptone,  and  this  may  be 
effected  by  a  large  number  of  different  bacteria.  The 
special  products  of  putrefaction  vary  according  to  the 


Highly  Organized  Flora  of  Running  Streams.  13. 

nature  of  the  material  and  the  conditions  in  which  it  is 
placed  and  the  micro-organisms  present.  One  or  other  of 
the  bacteria  concerned  will  take  precedence,  when  circum- 
stances favor  its  growth.  Thus,  the  aerobic  bacteria  can- 
not grow  unless  putrefying  material  is  freely  exposed  to 
atmospheric  oxygen.  The  anaerobic  bacteria  require  its 
exclusion.  Some  saprophytic  bacteria  grow  at  a  low  tem- 
perature ;  others  take  precedence  when  the  temperature  is 
high ;  some,  no  doubt,  thrive  only  in  the  presence  of  pro- 
ducts evolved  by  other  species  and  are  consequently  asso- 
ciated with  and  dependent  upon  these  species.  Some  are 
restrained  in  their  growth  sooner  than  others,  by  products 
evolved  as  the  result  of  their  own  vital  activity,  or  by  that 
of  associated  organisms.  Some  grow  in  the  presence  of 
acids,  and  give  rise  to  fermentation,  which  wholly  prevents 
the  development  of  their  species. 

The  malodorous  volatile  products  of  putrefaction  are  to 
a  considerable  extent  produced  by  the  anaerobic  species. 
For  this  reason  these  odors  are  more  pronounced  when 
masses  of  albuminous  material  undergo  putrefaction  in 
situations  where  the  oxygen  of  the  air  has  not  access,  or 
where  it  is  displaced  by  carbon  dioxide.  The  gases  pro- 
duced in  the  interior  of  a  putrefying  mass  are  mainly  CH4, 
H2S  and  H.  Many  of  the  bacteria  of  putrefaction  are 
facultative  anaerobics  ;  they  are  able  to  multiply  either  in 
the  presence  or  absence  of  oxygen.  The  products  formed 
by  these  differ,  no  doubt,  according  to  whether  they  are  or 
are  not  supplied  with  atmospheric  oxygen.  The  decom- 
position due  to  aerobic  bacteria  is  not  attended  with  the 
same  putrefaction  odors  as  in  the  case  of  the  anaerobic 
organisms ;  the  products  evolved  being  of  a  simple  chemi- 
cal composition, — CO2,  NH3. 

The  most  common  organisms  in  running  and  stagnant 
water  are  the  leptotricheae  and  the  cladotricheae.  The 
former  includes  the  four  genera :  Crenothrix,  Beggiatoa, 
Phragmidiothrix,  and  Leptothrix, — the  latter  a  single 


14        Biological  Relation  Between  Bacteria  and  the  More 

genus,  Cladothrix.  These  organisms  are  widely  distrib- 
uted, and  are  found  both  in  salt  and  fresh  water  containing 
decomposing  animal  or  vegetable  material ;  in  sulphurous 
waters  the  beggiatoa  are  especially  abundant  and  accumu- 
late upon  the  muddy  bottom,  or  upon  organic  substances 
undergoing  decomposition.  They  cover  the  bottom  of 
ponds  or  of  small  bays,  forming  different  colored  layers  of 
an  extended  and  abundant  growth.  These  organisms  are 
found  plentifully  in  the  refuse  waters  of  sugar  refineries 
and  upon  the  surface  of  putrefying  vegetable  and  animal 
material,  in  fresh  or  salt  waters.  The  cladothrix  dicho- 
mata  is  frequently  associated  with  the  beggiatoa  and  is 
common  in  the  refuse  water  of  factories,  especially  sugar 
factories.  It  may  readily  be  obtained  from  the  surface  of 
putrefying  algae  or  animal  substances  immersed  in  river 
or  swamp  water. 

As  far  as  can  be  determined  with  the  limited  experi- 
mental evidence  at  hand,  it  appears  evident  that  the  first 
stage  of  the  reduction  and  solution  of  vegetable  and  animal 
organic  matters  in  running  streams  is  traceable  to  the  single 
or  combined  action  of  the  crenothrix,  beggiatoa  and  cla- 
dothrix. After  these  organisms  have  in  part  broken  up 
the  organic  matters,  with  the  assistance  of  the  lower  forms 
of  animal  life  also  present  in  the  water,  the  subsequent 
stages  in  the  operation  are  traceable  to  the  action  of  a 
species  of  bacteria  of  which  the  nitronomas  of  Wino- 
gradsky  and  the  nitrifying  bacillus  of  Winogradsky  are, 
perhaps,  the  best-known  members.  These  organisms  in 
turn  attack  the  products  of  decomposition  produced  by 
the  beggiatoa  and  cladothrix,  and  convert  them  into  nitric 
acid.  These  nitrifying  bacteria,  as  they  are  called,  are 
the  principal  agents  concerned  in  the  reduction  of  organic 
matter  and  its  conversion  back  into  inorganic  matter. 
They  are  the  active  agents  upon  which  the  efficiency  of 
sand-filtration  in  the  purification  of  water  supplies  and  of 
sewage  is  dependent.  These  organisms  form  a  slimy  layer 


Highly  Organized  Flora  of  Running  Streams.  15 

composed  of  zoogloa  masses  of  bacilli  covering  the  sur- 
face of  the  filtering  bed  and  restrain  the  passage  of  all 
organic  matter,  both  living  and  dead,  and  break  it  up 
into  inorganic  salts. 

The  relation  of  these  lower  forms  of  micro-organisms, 
all  of  which  are  chlorophyl-free,  to  the  chlorophyl-bearing 
organisms  found  in  running  streams  is  a  most  important 
one.  Through  their  operation  the  organic  impurities  are 
broken  up  and  mineralized,  so  that  in  turn  they  can  again 
serve  as  food  material  for  the  chlorophyl-bearing  organisms. 
The  relation  of  these  two  groups  of  organisms  in  running 
streams  is  similar  to  that  of  these  organisms  when  found 
in  the  soil  and  elsewhere.  There  is  no  doubt  that  were 
all  of  the  chlorophyl-free  organisms  to  be  removed  from 
the  face  of  the  earth,  the  life  history  of  higher  vegetation 
would  be  a  comparatively  brief  one ;  being  dependent 
upon  the  continuous  supply  of  the  mineral  salts  capable 
of  serving  as  food  material  upon  the  activity  of  the 
chlorophyl-free  organisms.  As  soon  as  all  of  this  avail- 
able food  material  would  be  used  up  their  life  history 
would  be  completed,  and  in  turn  the  animal  world  would 
be  brought  to  the  same  termination  as  soon  as  all  vegetable 
life  had  ceased. 


COMPARATIVE  STUDIES 


UPON  THE 


PSEUDO-DIPHTHERIA,  OR  HOFMANN 
BACILLUS,  THE  XEROSIS  BACILLUS, 
AND  THE  LOFFLER  BACILLUS 


BY 


D.  H.  BERGEY,  M.  D.,  First  Assistant 

Laboratory  of  Hygiene,  University  of  Pennsylvania 


COMPARATIVE   STUDIES  UPON  THE    PSEUDO-DIPHTHERIA,  OR 

HOFMANN   BACILLUS,  THE  XEROSIS  BACILLUS,  AND 

THE  LOFFLER  BACILLUS. 

By  D.   H.    Bergey,   M.  D., 
First  Assistant,  laboratory  of  Hygiene,  University  of  Pennsylvania. 

The  group  of  pseudo-diphtheria  bacilli  and  the  xerosis 
bacillus  are  now  of  very  great  interest  because,  by  their 
marked  similarity  to  the  Loffler  bacillus  in  many  of  their 
morphological  and  biological  characters,  they  lead  to  un- 
certainty in  the  diagnosis  of  true  diphtheria  by  micro- 
scopic examination  alone.  The  pseudo-diphtheria  bacilli 
are  sometimes  found  in  cases  of  true  diphtheria  in  connec- 
tion with  the  Loffler  bacillus,  but  more  frequently  in  cases 
of  benign  throat  affections,  either  alone  or  in  connection 
with  staphylococci  and  streptococci.  The  xerosis  bacillus 
is  found  very  frequently  on  the  conjunctiva  of  healthy 
persons,  where  it  apparently  gives  rise  to  no  disturbances. 
It  is  also  found  in  the  nasal  cavity  and  the  throat  of  per- 
sons suffering  from  benign  inflammations  of  the  lining 
membrane  of  these  cavities.  Both  of  these  groups  of 
organisms  have  also  been  found  upon  the  skin  of  various 
portions  of  the  body,  without  at  times  apparently  pro- 
ducing any  appreciable  symptoms ;  at  other  times  they 
have  been  found  in  various  skin  diseases,  in  the  genito- 
urinary organs  of  healthy  persons,  as  well  as  in  various 
diseased  conditions  of  these  organs. 

The  pseudo-diphtheria  bacilli  were  first  described  by 
Loffler  (i)  and  by  von  Hofmann-Wellenhof  (2),  who  re- 
garded them  as  being  non-virulent  forms  of  the  Loffler 
bacillus. 

Roux  and  Yersin  (3)  demonstrated  that  the  Loffler  bacil- 
lus possessed  great  variations  in  virulence,  sometimes 
being  non-virulent,  and  they  were  also  of  the  opinion  that 

(19) 


2O  Comparative  Studies 

the  pseudo-diphtheria  bacilli  were  non-virulent  forms  of  the 
Loffler  bacillus.  Roux  and  Yersin  isolated  the  pseudo- 
diphtheria  bacillus  from  the  mucus  of  the  pharynx  and 
tonsils  of  children,  as  follows :  From  forty-five  children 
suffering  from  various  affections,  not  diphtheritic,  fifteen 
times ;  from  fifty-nine  healthy  children,  twenty-six  times. 
They  found  the  bacillus  in  five  out  of  seven  cases  of 
measles.  Inoculations  into  animals  never  produced  fatal 
results.  At  times  a  notable  edema  was  produced,  espe- 
cially so  with  cultures  obtained  from  cases  of  measles. 
They  state  that  the  organisms  can  only  be  differentiated 
from  the  Loffler  bacillus  by  inoculations  into  animals. 
Their  morphological  and  cultural  differences  prove  nothing. 

The  xerosis  bacillus  was  discovered  in  large  quantities 
by  Kuschbert  and  Neisser  (Breslau  artzliche  Zeitschrift, 
1883,  No.  4)  in  a  condition  known  as  xerosis  conjunctivae, 
and  their  observations  have  been  confirmed  by  many  others. 

A.  Neisser  (4)  reports  his  studies  on  what  he  calls  spore- 
formation  of  xerosis  bacilli  and  other  organisms.  He  states 
that  when  stained  nearly  all  the  bacilli  show  deeply-stained 
poles,  with  an  unstained  centre ;  sometimes  entire  separa- 
tion into  two  short,  almost  square  halves.  The  clear 
space  in  the  centre  is  not  a  spore. 

Ernst  (5)  isolated  the  xerosis  bacillus^from  a  twelve- 
year-old  boy  with  well-marked  xerosis  with  hernoralopie. 
He  describes  the  polar  granules  observed  in  these  organisms. 

Abbott  (6)  studied  the  occurrence  of  the  pseudo-diph- 
theria bacillus  in  benign  throat  affections,  such  as  acute 
pharyngitis,  follicular  tonsillitis,  post-nasal  catarrh,  simple 
enlargement  of  the  tonsils,  chronic  pharyngitis,  subacute 
and  chronic  laryngitis,  and  rhinitis.  Out  of  fifty-three 
patients  examined  forty-nine  presented  nothing  peculiar. 
A  variety  of  micro-organisms  were  found,  most  commonly 
the  pyogenic  cocci.  In  four  cases  micro-organisms  were 
found  which  resembled  the  L,6fHer  bacillus  morphologically, 
but  were  found  to  be  non-pathogenic. 


Upon  the  Pseudo- Diphtheria,  etc.  21 

Biggs,  Park  and  Beebe  (7)  reached  the  conclusion  that 
from  the  fact  that  there  is  no  means  of  determining  by 
cultural  methods  any  difference  between  the  Loffler  bacil- 
lus and  the  pseudo-diphtheria  bacillus,  and  that  the  only 
difference  noticeable  is  that  of  virulence,  the  pseudo- 
diphtheria  bacilli  are  non-virulent  L,6ffler  bacilli.  They 
found  some  of  the  cultures  to  produce  acid  in  glucose 
broth,  while  others  did  not.  The  fact  that  some  of  the 
micro-organisms  which  resemble  the  Loffler  bacillus  have 
acid  products  while  others  do  not,  together  with  other 
points  of  difference,  led  them  to  suggest  that  the  name 
pseudo-diphtheria  bacillus  had  been  applied  to  two  distinct 
organisms. 

Fraenkel  (8)  reports  on  the  examination  of  a  number  of 
organisms  isolated  in  various  forms  of  disease  of  the  con- 
junctiva, as  acute  and  chronic  conjunctivitis,  trachoma, 
and  from  the  healthy  conjunctiva.  Among  the  organisms 
isolated  there  were  two  derived  from  cases  of  xerosis  con- 
junctivae.  He  is  inclined  to  the  opinion  that  the  organ- 
isms described  as  xerosis  bacilli  by  Kuschbert  and  Neisser 
(Deutsche  Medicinische  Woch.,  1884  ;)  Fraenkel  and  Franke 
(Archiv  fur  Augenheilkunde,  Bd.  7,  1887) ;  Schreiber 
(Fortschritte  der  Medicin,  1888,  p.  650)  ;  Brnst  (5),  and 
others,  were  pseudo-diphtheria  bacilli.  He  is  brought  to 
this  opinion  by  the  fact  that  we  find  in  cases  of  true  diph- 
theria both  virulent  bacilli  and  others  whose  virulence  has 
been  weakened  or  lost ;  also  by  the  fact  that  this  is  true  of 
a  large  number  of  other  organisms,  as  the  pneumococcus, 
streptococcus  pyogenes,  bacillus  coli  communis,  and  others. 
He  believes  that  it  is  not  unlikely  that  the  so-called  xerosis 
bacilli  of  Neisser  are  nothing  less  than  true  diphtheria 
bacilli  robbed  of  their  virulence. 

There  is  some  difference  of  opinion  as  to  the  xerosis 
bacillus  occurring  in  normal  conjunctival  secretions.  On 
the  one  hand  Uthoft  (9)  states  that  it  is  frequently  pres- 
ent in  normal  eyes.  He  treated  a  child  with  conjunc- 


22  Comparative  Studies 

tivitis  crouposa  for  fourteen  days.  A  culture  taken  from 
the  conjunctiva  was  identical  with  the  Loftier  bacillus. 
Fourteen  days  after  recovery  the  child  was  again  examined 
and  a  culture  obtained  from  the  conjunctiva  which  was 
identical  with  the  first,  except  that  it  was  non-virulent. 
These  cultures  were  examined  by  Fraenkel  and  showed 
the  same  results.  On  the  other  hand,  Franke  (10)  examined 
128  normal  conjunctivae  without  being  able  to  discover 
the  bacillus. 

Abbott  (n)  in  a  paper  on  the  etiology  of  membranous 
rhinitis  states  his  conclusions  as  follows  : 

"  We  are  inclined  to  the  opinion  that  the  term  '  pseudo- 
diphtheritic  bacillus '  as  applied  to  an  organism  in  all  re- 
spects identical  with  the  genuine  diphtheritic  bacillus, 
save  for  its  inability  to  kill  guinea-pigs  when  inoculated 
subcutaneously,  is  a  misnomer,  and  that  it  would  be  more 
nearly  correct  to  designate  this  organism  as  the  attenuated 
or  non-virulent  diphtheritic  bacillus,  reserving  the  term 
*  pseudo-diphtheritic '  for  that  organism  or  group  of  or- 
ganisms (for  there  are  probably  several)  that  is  enough  like 
the  diphtheria-bacillus  to  attract  attention,  but  is  dis- 
tinguishable from  it  by  certain  morphological  and  cultural 
peculiarities  aside  from  the  question  of  virulence." 

Howard  (12)  found  the  organism  in  ulcerative  endocardi- 
tis, both  in  the  valves  and  in  other  internal  organs.  Bern- 
heim  (13)  in  one  case  of  diphtheria  found  also  the  pseudo- 
diphtheria  bacillus,  which  proved  to  be  non-pathogenic. 
He  noticed  no  necrosis  nor  loss  in  body-weight  in  the  ani- 
mals inoculated,  thus  differentiating  between  the  pseudo- 
diphtheria  bacillus  and  diphtheria  bacilli  that  are  of  low 
virulence.  Davolos  (14)  found  the  bacillus  in  a  case  of 
impetigo.  Deyl  (15)  isolated  it  in  fifteen  cases  of  "  chala- 
zion ' '  formation,  and  considered  that  it  was  the  cause  of 
the  disease.  He  was  able  to  produce  such  formations  in 
animals,  by  means  of  inoculation  with  this  organism.  He 
also  found  the  organisms  in  gonorrhoea  and  blenorrhoea. 


Upon  the  Pseudo- Diphtheria,  etc.  23 

It  has  been  isolated  in  other  situations:  for  instance, 
Neisser  states  that  he  found  it  in  cases  of  vaginal  discharge, 
ulcers  of  the  leg,  etc.  Kruse  and  Pasquale  (16),  in  a  case 
of  liver  abscess  following  dysentery,  found  a  bacillus  which 
they  named  bacillus  clavatus,  that  appeared  in  every  respect 
to  resemble  the  pseudo-diphtheria  bacillus.  It  was  non- 
pathogenic  for  guinea  pigs.  Babes  (17)  found  it  in  eight 
cases  of  trachoma  and  in  a  case  of  gangrene  of  the  lung. 
Bass  (18)  found  it  in  two  cases  of  chronic  liver  disease 
accompanied  with  disturbed  vision,  resembling  hemoralo- 
pie  and  xerosis  conjunctivae.  The  xerosis  bacillus  was 
isolated  from  the  secretions  of  the  conjunctival  sac. 

Schanz  (19)  concludes,  from  the  results  of  his  investi- 
gation, that  the  xerosis  bacillus  differs  from  the  diphtheria 
bacillus  in  lack  of  virulence,  and  that  the  occurrence  of 
the  xerosis  bacillus  in  healthy  eyes  may  lead  to  diphtheria 
in  passing  from  the  tear-ducts  into  the  upper  air  passages, 
where  in  some  unknown  manner  it  acquires  virulence. 

Gerber  and  Podack  (20)  believe  that  it  is  not  impossible 
for  the  existence  of  some  relationship  between  pseudo- 
diphtheria  bacilli  and  diphtheria  bacilli,  in  cases  of  rhinitis 
fibrosa. 

Peters  (21)  found  that  the  differentiation  between  the 
xerosis  bacillus  and  the  pseudo-  and  true  diphtheria  bacilli 
cannot  be  made  from  their  morphological  and  cultural 
characters.  He  isolated  sixteen  cultures :  six  from  cases 
of  true  diphtheria,  three  from  cases  of  endemic  impetig- 
inous  eczema  of  the  face,  two  from  old  cases  of  recurring 
conjuctivitis  granulosa,*and  one  each  from  cases  of  xerosis 
conjunctivitis  crouposa,  pseudo-diphtheria  of  the  nose,  and 
from  the  healthy  mucous  membrane  of  the  nose. 

Spronck  (22)  sought  to  differentiate  between  the  xerosis 
and  pseudo-diphtheria  bacilli  and  Loffler  bacilli  by  means 
of  antitoxin.  He  states  that  the  antitoxin  prevented  the 
reaction  of  true  diphtheria  bacilli,  but  that  the  xerosis 
bacilli  were  still  capable  of  producing  local  symptoms, 


24  Comparative  Studies 

such  as  edema  and  inflammation,  even  more  so  than  when 
inoculated  alone.  It  also  produced  loss  of  appetite, 
drowsiness,  etc.,  in  the  animal  inoculated.  He  inoculated 
guinea  pigs  with  diphtheria  antitoxin,  and  subsequently 
inoculated  some  with  the  pseudo-diphtheria  bacilli  and 
others  with  the  true  diphtheria  bacillus.  Those  inoculated 
with  diphtheria  bacilli  remained  well,  while  those  inocu- 
lated with  the  pseudo-diphtheria  bacilli  showed  the  local 
reactions  observed  after  inoculations  with  these  organisms. 
He  believes  this  to  be  a  reliable  method  of  differentiating 
between  the  two  organisms. 

C.  Fraenkel  (23),  in  the  differentiation  between  the  true 
and  pseudo-diphtheria  bacilli,  also  experimented  with 
diphtheria  antitoxin,  free  from  antiseptic  material,  to 
determine  whether  it  could  serve  as  a  means  to  differentiate 
between  the  Loffler  bacillus  and  the  pseudo-diphtheria 
bacillus.  He  experimented  with  seven  cultures  of  the 
pseudo-diphtheria  bacillus  derived  from  the  eyelids  of 
healthy  persons,  and  from  several  cases  of  conjunctivitis 
crouposa  ;  but  the  results  were  entirely  negative.  He  then 
repeated  the  experiments  of  Spronck,  and  obtained  similar 
results.  Variable  quantities  of  the  cultures  were  injected 
into  the  subcutaneous  tissues  of  guinea  pigs,  in  this  manner 
establishing  the  character  of  the  cultures.  In  the  second 
series  of  experiments  he  also  used  antitoxin  at  the  same 
time.  The  results  were  as  follows :  Some  of  the  cultures 
he  injected  into  animals,  in  quantities  of  5  to  10  c.  c.,  pro- 
duced considerable  disturbance  at  the  point  of  injection  in 
the  nature  of  swelling  and  infiltration,  which  after  two 
days,  or  at  the  most  three  days,  entirely  disappeared.  In 
no  instance  did  he  observe  disturbances  of  general  nature 
reported  by  Spronck — as,  loss  of  appetite,  weakness,  loss  of 
weight,  or  change  in  temperature.  Even  in  very  large 
doses,  up  to  50  c.  c.,  the  results  were  approximately  the 
same.  There  were  no  fatal  results ;  these  results  indicat- 
ing that  the  antitoxin  was  without  influence.  The  result 


Upon  the  Pseudo- Diphtheria,  etc.  25 

was  the  same  where  the  antitoxin  serum  was  injected 
before,  at  the  same  time,  or  after  the  injection  of  the 
culture.  He  again  states  his  belief  that  it  is  not  unlikely 
that  the  so-called  xerosis  bacilli  are  nothing  else  than  true 
diphtheria  bacilli  robbed  of  their  virulence. 

Eyre  (24)  sums  up  his  investigations  by  stating  that  "  in 
differentiating  the  xerosis  bacillus  from  the  L,6ffler  bacillus 
we  are  saved  all  trouble  in  the  case  of  first  cultures  by  the 
fact  that  the  former  does  not  grow  on  blood  serum  at  37°  C. 
under  thirty-six  to  forty-eight  hours,  whilst  the  latter  makes 
its  appearance  in  eighteen  to  twenty-four  hours.  At  the 
other  extreme,  with  cultures  some  fifteen  to  twenty  gen- 
erations old,  there  is  likewise  very  little  difficulty  in  dis- 
tinguishing between  these  two  organisms,  as  the  xerosis 
bacillus  then  appears  as  a  much  shorter,  more  slender  and 
more  curved  bacillus,  exhibiting  neither  segmentation  nor 
clubbing.  But  in  the  case  of  early  sub-cultures  from  the 
first  culture,  the  circumstances  are  entirely  altered,  and  we 
have  to  deal  with  an  organism  closely  resembling  in  its 
general  characters  and  mode  of  growth  the  L,6fHer  bacillus 
— an  organism  moreover  which  has  no  one  single  persistent 
peculiarity  which  will  enable  us  to  say  definitely,  this  is 
the  xerosis  bacillus.  We  have  therefore  to  depend  upon 
the  sum  total  of  the  cultural  and  morphological  differences 
— minute  in  themselves — picked  out  during  the  course  of 
numerous  observations. 

As  to  the  exact  nature  of  the  xerosis  bacillus — whether 
it  be  a  non-virulent  and  slightly  altered  species  of  the 
bacillus  diphtheriae  or  a  totally  separate  and  distinct 
bacillus — it  is  impossible  at  present  to  decide. 

Schanz  (25)  reports  that  in  ten  normal  conjunctival  sacs 
he  found  the  xerosis  bacillus  four  times ;  finding  it  to  be 
the  most  frequent  organism  of  the  conjunctival  sac.  In 
comparing  one  derived  from  a  case  of  xerosis  with  kera- 
tomalacia  with  the  Loffler  bacillus,  he  found  that  a  certain 
differentiation  of  the  two  organisms  could  not  be  made,  on 


26  Comparative  Studies 

the  ground  of  their  cultural  and  morphological  characters. 
He  states  that  the  lack  of  virulence  on  the  part  of  the 
xerosis  bacillus  cannot  be  taken  as  a  differentiating  feature, 
because  the  true  diphtheria  bacillus  is  also  at  times  non- 
virulent.  He  believes  the  xerosis  bacillus  to  be,  as  long 
as  no  further  point  of  differentiation  is  known,  a  diphtheria 
bacillus  of  low  virulence,  and  holds  it  to  be  identical  with 
the  pseudo-diphtheria  bacillus. 

The  Massachusetts  State  Board  of  Health  (26)  reports 
on  some  cultures  isolated  in  the  routine  examination  of 
cultures  taken  from  suspected  cases  of  diphtheria.  "Four 
of  the  forty-six  cultures  isolated  were  found  to  be  pseudo- 
diphtheria  bacilli.  A  few  cylindrical-,  pear-  and  hour-glass 
shaped  bacilli  are  occasionally  seen,  but  involution  forms 
are  not  marked,  even  in  old  cultures.  They  are  distin- 
guished from  diphtheria  bacilli  by  being  shorter,  smaller, 
more  uniform  in  size,  shape  and  manner  of  staining  and, 
as  pointed  out  by  Escherich,  by  a  tendency  to  lie  parallel 
in  cover-slip  preparations.  These  bacilli  are  of  occasional 
occurrence,  both  in  the  throats  of  persons  suffering  from 
non-diphtheritic  throat  affections  and  in  true  diphtheria 
mingled  with  the  Loffler  bacillus.  It  is  only  in  convalescent 
cases  of  long  duration  that  the  pseudo-diphtheria  bacilli 
are  likely  to  cause  doubt.  They  might  be  mistaken  for 
the  last  few  remaining  diphtheria  bacilli,  or  the  reverse 
might  occur.  A  few  remaining  virulent  forms  may  be 
regarded  as  pseudo  forms.  Diphtheria  bacilli  directly 
from  the  membrane  of  the  throat  or  from  cultures  scarcely 
developed  sometimes  resemble  quite  closely  the  pseudo- 
diphtheria  bacilli  in  morphology  and  staining. 

"The  morphological  differences  are  reinforced  by  at 
least  two  biological  differences  of  importance — the  absence 
of  any  power  to  produce  acids  in  bouillon  containing 
dextrose,  and  the  lack  of  pathogenic  power. 

"Though  there  are  these  three  distinctive  features 
of  pseudo-diphtheria  bacilli — characteristic  morphology> 


Upon  the  Pseudo- Diphtheria,  etc.  27 

absence  of  acid  and  of  toxic  production — it  is  not  a  simple 
matter  to  recognize  them  as  such  promptly  under  the 
microscope  when  taken  from  throat  cultures,  unless  the 
observer  has  had  considerable  training.  It  is  highly  prob- 
able, therefore,  that  Roux  and  Yersin  in  their  earlier  work 
may  have  mistaken  pseudo-diphtheria  for  true  diphtheria 
bacilli,  when  they  found  virulent  and  non-virulent  forms 
together  in  the  throats  of  convalescents.7' 

E.  A.  Peters  (27)  states  that  the  relation  of  the  Hofmann 
bacillus  to  the  true  diphtheria  bacillus  is  clear  on  certain 
points : 

(a)  "The    Hoffmann   bacilli   resemble   the    diphtheria 
bacilli  in  mode  of  growth,  and  slightly  in  microscopical 
character. 

(b)  "The  cases  in  which  they  are  found  are  liable  to  be 
mistaken    for   mild   diphtheria.     The  prognosis   in   such 
cases  is  good. 

(c)  "  There  is  no  proof  forthcoming  that  this  bacillus  is 
an  attenuated  form  of  the  diphtheria  bacillus ;  though  the 
short  diphtheria  bacillus,  when  it  becomes  non-pathogenic, 
tends  to  resemble  the  Hofmann  bacillus." 

Schanz  (28)  employed  as  a  differential  diagnostic  method 
tests  of  the  acidity  of  the  bouillon  cultures.  Earlier  in- 
vestigators have  shown  that  the  pseudo-diphtheria  bacilli 
are  also  able,  like  the  diphtheria  bacilli,  to  produce  acid. 
Neisser  was  able  to  substantiate  this,  yet  the  amount  of 
acid  with  the  pseudo-diphtheria  bacilli  after  growing  for 
forty-eight  hours  was  from  four  to  five  times  smaller  than 
with  the  diphtheria  bacilli.  Schanz  found  only  one  excep- 
tion, with  a  xerosis  culture,  which  showed  an  equal  acidity 
with  that  found  in  the  diphtheria  culture.  The  amount 
of  acid  formed,  he  says,  is  dependent  to  some  extent  upon 
the  amount  of  the  culture  inoculated  into  a  tube. 

Trumpp  (29)  differentiated  between  the  diphtheria  and 
pseudo-diphtheria  bacilli  by  means  of  simultaneous  inocu- 
lations of  diphtheria  toxin.  The  amount  of  toxin  inocu- 


28  Comparative  Studies 

lated  was  less  than  the  minimum  fatal  dose,  so  that  the 
additional  toxin  generated  by  the  diphtheria  bacilli  of  low 
virulence  would  be  sufficient  to  kill  the  animal.  Control 
animals  inoculated  with  like  amounts  of  toxin  remained 
alive.  He  believes  that  it  is  not  impossible  to  increase  the 
virulence  of  acid-producing  bacilli,  but  that  non-acid  pro- 
ducing pseudo-diphtheria  bacilli  cannot  be  made  virulent. 
He  succeeded  in  making  a  non-virulent  bacillus,  derived 
from  a  case  of  empyema  following  measles,  virulent  by 
inoculating  it  along  with  diphtheria  toxin. 

Prochaska  (30)  reports  on  sixteen  cases  in  which  the 
pseudo-diphtheria  bacilli  were  found.  These  cases  were 
found  in  making  diagnoses  of  diphtheria,  and  were  all 
cases  of  throat  infection.  Thirteen  of  the  cultures  were 
from  cases  of  follicular  angina.  Another  culture  came 
from  a  case  of  pharyngeal  and  nasal  diphtheria.  In  this 
case  the  typical  L,6fner  bacilli  were  also  found.  When 
grown  on  blood  serum  they  could  not  be  differentiated  from 
Loffier  bacilli  by  their  size,  and  on  microscopical  examina- 
tion resembled  the  pseudo-diphtheria  bacilli.  Inoculation 
into  animals  gave  negative  results.  The  other  two  cultures 
were  isolated  from  the  throats  of  children  sick  with  scarlet 
fever.  In  each  of  these  cases  there  was  a  typical  diphtheria 
membrane  in  the  throat,  yet  no  virulent  bacilli  could  be 
found. 

M.  Neisser  (31)  gives  a  method  of  staining  for  differ- 
entiating between  the  diphtheria  bacilli  and  pseudo- 
diphtheria  bacilli  which  he  considers  very  reliable.  The 
solutions  used  are,  first,  one  gramme  powdered  methylene 
blue  (Griibler)  dissolved  in  20  c.  c.  of  96  per  cent  alcohol ; 
to  this  is  added  950  c.  c.  of  distilled  water  and  50  c.  c.  of 
acetic  acid;  and,  second,  two  grammes  of  vesuvin  dis- 
solved in  a  litre  of  boiled  distilled  water.  Filtration, 
especially  of  the  latter  solution,  is  necessary.  Cover-slips 
prepared  in  the  usual  manner  are  stained  in  the  first  solu- 
tion for  from  one  to  three  seconds,  washed  in  water,  and 


Upon  the  Pseudo- Diphtheria,  etc.  29 

stained  for  from  three  to  five  seconds  in  vesuvin,  washed  in 
water  and  examined.  This  process  will  show  beautiful 
double  staining  of  the  diphtheria  bacilli,  when  grown  on 
LofHer's  blood  serum  for  ten  to  twenty  hours  at  34°  to 
35°  C.,  while  the  pseudo-diphtheria  bacilli  show  no  double 
staining.  Neisser  believes  it  would  be  best  to  drop  the 
name  pseudo-diphtheria,  and  "designate  as  pseudo-diph- 
theria bacilli  only  those  described  by  von  Hofmann  and 
Loftier.  The  only  difficulty  in  differentiation  is  with  the 
group  of  xerosis  bacilli  and  a  group  of  rather  thick,  short 
strepto-bacilli."  He  reports  on  a  careful  study  of  twenty-two 
bacilli  resembling  the  lyofHer  bacillus.  Some  of  these  were 
derived  from  the  throat,  from  the  nose  in  nasal  diphtheria 
and  from  the  conjunctiva.  When  grown  on  blood  serum 
the  growth  of  the  strepto-bacilli  was  observed.  But  the 
similarity  between  the  two  is  less  marked,  and  there  is  an 
absence  of  the  longer  forms  of  the  bacilli.  When  stained 
by  Gram's  method  they  appear  larger,  and  may  lead  to 
uncertainty.  The  xerosis  bacilli  show  very  slight  growth 
after  six  hours.  When  stained  the  bacilli  appear  older 
than  the  diphtheria  bacilli  of  the  same  age.  The  growth 
of  the  xerosis  bacilli  after  ten  hours  is  still  not  very 
marked.  The  picture  which  they  present  is  sometimes 
quite  similar  to  that  presented  by  the  diphtheria  bacilli, 
the  form  altogether  so ;  yet  not  like  the  diphtheria  bacilli 
of  this  age  usually  appear  in  such  cultures.  When  studied 
with  the  method  of  double  staining  which  he  describes  it 
is  only  in  sixteen-  to  twenty-hour-old  cultures  that  it 
becomes  possible  to  make  a  differential  diagnosis;  the 
pseudo-diphtheria  bacilli  showing  a  negative  appearance, 
like  most  of  the  xerosis  forms.  At  times,  however,  there 
are  single  individuals  that  take  on  the  characteristic  stain- 
ing, but  it  is  impossible  to  mistake  the  picture  presented 
by  them  for  that  of  the  diphtheria  bacilli.  Cultures  several 
days  old  (this  is  especially  true  of  many  of  the  xerosis 
forms)  take  on  the  double  staining. 


30  Comparative  Studies 

Olmacher  (32)  found  the  pseudo-diphtheria  bacilli  in  a 
case  of  pneumonia,  accompanied  with  purulent  meningitis. 

Cobbett  and  Phillips  (33)  state  that  uthe  frequent  occur- 
rence in  the  mouths  of  healthy  persons  of  organisms  which 
more  or  less  resemble  the  diphtheria  bacillus  makes  it 
desirable  that  we  should  have  some  test  which  would 
enable  us  to  distinguish  them  from  the  latter.  A  satis- 
factory test  of  this  kind  has  indeed  not  yet  been  found. 
We  know  of  no  other  way  of  distinguishing  the  non- 
virulent  acid-producing  bacillus  but  by  the  injection  of 
animals.  On  the  other  hand,  the  cultivation  of  a  suspected 
diphtheria  bacillus  in  an  alkaline  glucose  broth  gives  us  a 
ready  means  of  excluding  the  other  simulator  of  the  diph- 
theria bacillus.  This  non-virulent  organism,  which  is 
characterized  by  its  inability  to  produce  an  acid  reaction  in 
such  a  medium,  is  found  in  the  mouths  of  persons  suspected 
of  having  diphtheria  at  least  as  frequently  as  is  the  non- 
virulent  acid  producer.  We  therefore  venture  to  recom- 
mend that  this  simple  test  be  applied  to  any  organism 
suspected  of  being  the  lyoffler  bacillus." 

Fraenkel  (34)  investigated  the  value  of  Neisser's  special 
method  of  differential  staining,  and  says  that  when  the 
conditions  laid  down  by  Neisser  are  strictly  observed  in 
every  particular  this  method  affords  a  highly  satisfactory 
diagnostic  method.  He  found  three  cultures  which  gave 
a  distinct,  positive  result  with  this  special  staining,  and 
also  rendered  the  culture  media  acid,  and  in  other  respects 
resembled  the  true  diphtheria  bacilli,  except  that  they 
were  non-pathogenic  for  guinea  pigs,  in  very  large  doses. 
Two  of  these  cultures  were  derived  from  cases  of  typical 
diphtheria,  and  he  is  undecided  whether  these  are  non- 
virulent  Loffler  bacilli  or  not. 

Zupink  (35)  in  a  comparative  investigation  of  some 
pseudo-diphtheria  bacilli  found  the  Gram  method  of  stain- 
ing was  of  some  value  in  differentiating  between  these 
organisms  and  the  LofHer  bacillus.  He  divides  the  organ- 


Upon  the  Pseudo- Diphtheria,  etc.  31 

isms  into  the  group  of  pseudo-diphtheria  bacilli  and  the 
Loffler  group. 

De  Martini  (36)  experimented  with  two  cultures  of  the 
pseudo-diphtheria  bacillus,  the  one  rendering  neutial  bouil- 
lon acid,  the  other  rendering  it  alkaline.  The  former  did 
not  grow  in  fluid  diphtheria  antitoxin,  while  the  latter 
grew  well,  while  both  grew  as  well  on  coagulated  antitoxin 
as  on  ordinary  coagulated  blood  serum.  The  L,6ffler  ba- 
cillus did  not  grow  well  in  the  fluid  antitoxin,  but  grew 
well  after  coagulation.  He  believes  that  the  acid-produc- 
ing pseudo-diphtheria  bacillus  is  a  degenerated  Loffler 
bacillus,  while  the  non-acid  producing  bacillus  is  a  differ- 
ent form  of  organism.  From  his  studies  he  is  inclined  to 
the  opinion  that  we  are  not  dealing  with  simply  modifica- 
tions or  varieties  of  the  same  form,  but  with  forms  of 
bacilli,  each  having  its  own  identity. 

Spronck  (37)  in  a  lengthy  dissertation  touches  again  on 
the  question  previously  discussed  by  him, — on  the  differen- 
tiation of  the  true  and  pseudo-diphtheria  bacillus.  He 
states  that  the  difference  in  growth  claimed  by  Martini 
and  Nicolas  in  diphtheria  antitoxin  was  not  observed  by 
him,  wherein  his  results  coincide  with  those  of  C.  Fraen- 
kel,  though  he  found  diphtheria  bacilli  that  grew  poorly 
on  the  serum,  but  there  were  others  that  grew  without 
difficulty.  As  the  only  method  of  establishing  the  true 
character  of  a  doubtful  culture,  he  again  advises  the 
use  of  the  inoculation  method  with  the  serum.  The 
pseudo-bacilli  produce  a  swelling  of  the  subcutaneous 
tissues  in  large  doses ;  even  when  previously  injected 
with  serum,  the  true  bacillus  is  without  influence. 

Fraenkel  (38)  says  that  this  tedious  method  of  differen- 
tiation is  now  no  longer  necessary  since  the  introduction 
of  the  method  of  double-staining  by  M.  Neisser.  He  has 
during  the  past  few  months  used  Neisser's  method  on  a 
large  number  of  true  and  pseudo-diphtheria  bacilli,  and  is 
able  to  substantiate  the  statements  of  Neisser.  Likewise, 


32  Comparative  Studies 

it  can  be  stated  with  safety  that  a  culture  which  fails  to 
give  the  granule  formation  under  the  proper  conditions 
is  not  a  culture  of  the  true  diphtheria  bacillus  ;  on  the 
other  hand,  a  culture  which  gives  a  positive  result  is  most 
probably  not  pseudo-diphtheria.  He  is  not  convinced 
that  his  former  opinion  as  to  the  identity  of  the 
two  groups  is  erroneous,  and  that  we  have  to  deal 
with  two  distinct,  if  very  closely  related,  forms  of  bac- 
teria. 

Muir  and  Ritchie  (39)  state  that  "  the  term  xerosis 
bacillus  has  been  given  to  an  organism  first  observed  by 
Kuschbert  and  Neisser  in  xerosis  of  the  conjunctiva,  and 
which  has  since  been  found  in  many  other  affections  of  the 
conjunctiva,  and  even  in  normal  conditions.  Morphologi- 
cally, it  is  practically  similar  to  the  diphtheria  bacillus, 
and  even  in  cultures  presents  very  minor  differences.  It 
is,  however,  non-virulent  to  animals  and,  according  to 
Eyre,  does  not  produce  an  acid  reaction  in  neutral  bouil- 
lon ;  in  this  way  it  can  be  distinguished  from  the  diph- 
theria bacillus." 

Gliicksmann  (40)  states  that  the  pseudo-diphtheria  bacilli 
are  shorter  and  more  plump  than  the  true  diphtheria  bacillir 
mostly  pointed  at  both  ends,  wedge-shaped  ;  less  frequently 
they  are  club-shaped.  In  the  children's  hospital  he  found, 
in  cases  which  showed  no  membrane,  and  had  no  fever, 
that  the  pseudo-diphtheria  bacillus  was  present  in  twenty 
out  of  thirty-nine  cases  examined.  L,arge  quantities  of  a 
culture  of  the  pseudo-diphtheria  bacillus  were  inoculated 
into  guinea  pigs  without  producing  any  effects,  except  at 
times  a  slight  local  infiltration.  The  pseudo-diphtheria 
bacillus  was  found  not  to  immunize  the  guinea  pigs  against 
the  diphtheria  bacillus.  An  animal  inoculated  twenty 
times  with  large  doses  (15  c.  c.)  of  a  culture  failed  to  show 
any  immunization,  as  it  died  promptly  when  inoculated 
with  diphtheria.  He  believes  that  inoculation  into  ani- 
mals is  the  only  mode  of  differentiation. 


Upon  the  Pseudo- Diphtheria,  etc.  33 

Axenfeld  (41)  notes  certain  differences  between  these 
two  groups  of  bacilli  when  grown  on  agar,  bouillon  and 
blood  serum.  First,  the  so-called  xerosis  bacillus  grows 
sparingly  on  the  agar,  very  slowly,  sometimes  showing 
only  after  three  or  four  days.  It  shows  as  small,  very  dry, 
closely-adherent  colonies  ;  while  the  Hofmann-Loffler  pseu- 
do-diphtheria bacillus  grows  more  rapidly,  in  thicker  lay- 
ers, moist,  glistening,  and  easily  removed.  In  bouillon  the 
xerosis  bacillus  produces  no  clouding,  with  only  small 
flakes  at  the  bottom.  The  alkalinity  is  not  increased. 
The  Hofmann-Loffler  bacillus  grows  as  distinct,  slimy 
substances  at  the  bottom  of  the  tube,  with  rapid  clouding 
of  the  bouillon  and  increase  of  the  alkalescence.  On  blood 
serum  the  xerosis  bacillus  grows  more  rapidly  than  on 
agar,  but  still  slowly,  forming  dry  colonies.  The  Hof- 
mann-Loffler bacillus  grows  much  more  rapidly  and  with  a 
more  moist  growth,  only  slightly  slower  than  the  diphtheria 
bacillus.  He  says  that  so  far  attempts  have  been  unsuc- 
cessful to  change  the  form  of  one  of  these  growths  into 
the  other  through  long-continued  cultivation,  and  it  is 
impossible  to  speak  of  entire  identity  ;  yet  he  holds  it  as 
not  impossible  that  such  identity  may  in  time  be  demon- 
strated. He  is  inclined  to  believe,  contrary  to  Schanz, 
that  he  was  dealing  with  two  forms  of  one  family  of  organ- 
isms. 

Heimersdorff  (42)  investigated  the  value  of  Neisser's  dif- 
erential  staining  method  for  the  rapid  diagnosis  of  diph- 
theria of  the  conjunctiva.  At  the  University' eye  clinic, 
at  Breslau  and  Rostock,  all  cases  of  conjunctivitis  crouposa 
are  tested  according  to  the  Neisser  method.  He  has  found 
the  test  most  reliable  when  employed  with  fresh  cultures 
nine  to  sixteen  hours  old.  In  order  to  obtain  a  positive 
diagnosis  he  advises  the  simultaneous  inoculation  into 
animals.  So  far  the  diagnoses  made  by  the  Neisser 
method  of  staining  were  substantiated  by  the  inoculation 
tests.  He  calls  attention  to  the  importance  of  having  a 


34  Comparative  Studies 

proper  blood-serum  culture  medium  on  which  to  grow  the 
cultures,  and  advises  comparing  all  cultures  with  a  cul- 
ture of  the  L/offler  bacillus  grown  on  the  same  culture  me- 
dium, under  the  same  conditions. 

He  found  that  at  times  the  polar  granules  of  the  diph- 
theria bacillus  appear  later  than  sixteen  hours,  and  with 
the  xerosis  bacilli  earlier  than  twenty-four  hours,  and  states 
that  repeated  examinations  may  be  necessary  to  make  a 
positive  diagnosis. 

Kruse  (43)  under  bacillus  pseudo-diphthericus  (Pseudo- 
diphtherie,  Xerose  bacillus),  classes  bacilli  which  resemble 
the  diphtheria  bacillus,  but  are  not  pathogenic.  Besides 
the  studies  already  reported,  he  quotes  Zarinke  (^Central- 
blatt  fur  Bacteriologie,  Bd.  VI,  s.  6),  Beck  (Zeitschr.  fur 
Hygiene,  Bd.  VIII),  N.  Klein  (Centralblatt  fur  Bacteria- 
logie,  Bd.  VII,  s.  16),  Goldscheider  (ref.  Baumgarten's 
Jahrbericht.  92)  Bscherich  (Diphtheric,  Wien,  94),  Pflugger 
(Archiv  fur  Ophthal.,  37),  Kick  (Micro-organismen  in  Kon- 
junctivalsack,  Wiesbaden,  87),  who  also  report  on  studies 
on  these  organisms,  which  occur  in  30  per  cent  to  60  per 
cent  of  the  mouths  and  noses  of  all  persons,  but  more 
frequently  still  on  the  normal  or  diseased  conjunctiva. 
They  occur  in  great  masses  in  xerosis  conjunctiva. 

Kruse  places  in  this  group  also  the  bacillus  striatus 
albus  isolated  by  v.  Besser  from  the  nose  of  healthy  per- 
sons. Likewise  the  bacilli  found  by  Wilde  (Bonn  Hyg. 
Institui)  in  the  secretion  of  a  case  of  ozena,  and  the  organ- 
ism found  by  Ortman  (Berlin  klin.  Wochenschrift,  89)  in 
a  diphtheritic  deposit  on  the  mucous  membrane  of  the 
vaginae  of  pregnant  women,  and  those  found  by  Rugen- 
berg  (Bonn  Hyg.  Institut)  in  four  cases  of  impetigo.  He 
states  that  possibly  the  bacillus  nodosus  parvus  which 
lyustgarten  isolated  from  the  urethra  belongs  to  this 
group,  or  is  identical.  Also,  bacillus  endocarditis  griseus 
of  Weichselbaum,  and  bacillus  erythematis  uroglini  of 
Demme. 


Upon  the  Pseudo- Diphtheria ,  etc.  35 

Schiiltz  (44)  investigated  the  mixed  infections  in  pul- 
monary tuberculosis,  and  found  in  fifteen  out  of  thirty 
cases  examined  bacilli  resembling  the  diphtheria  bacillus. 
On  examining  these  cultures  when  grown  on  the  differ- 
ent culture  media,  he  found  an  almost  complete  identity 
with  the  Iy6fHer  bacillus,  though  nearly  every  culture  had, 
on  one  or  the  other  culture  medium,  a  slight  character- 
istic, either  in  the  form  or  size  of  the  individuals,  or  of 
the  culture  in  general.  Some  of  the  cultures  were  found 
to  be  pathogenic  for  guinea  pigs.  The  lesions  found 
after  death  were  injections  around  point  of  inoculation, 
exudate  into  the  internal  body  cavities,  and  hemorrhagic 
condition  of  the  adrenals.  Other  animals  died  after  longer 
intervals  without  any  lesions  indicative  of  diphtheria, 
only  cachexia  with  slight  peritonitis.  The  remaining 
cultures  were  found  to  be  non-pathogenic. 

Ehret  (45)  reports  on  five  cases  of  diabetic  tuberculosis 
in  which  he  also  found  the  pseudo-diphtheria  bacillus. 

Auckenthaler  (46)  investigated  the  value  of  Neisser's 
method  of  staining  in  the  diagnosis  of  diphtheria.  He 
found  that  whenever  LofHer  bacilli  were  present  in  the 
cultures  it  was  always  possible  to  demonstrate  the  polar 
granules  by  Neisser's  method.  He  believes  it  would  be 
better  to  allow  ten  to  [fifteen  seconds  for  the  operation  of 
the  methylene-blue  stain,  in  preference  to  one  to  three  sec- 
onds as  recommended  by  Neisser.  He  states,  however, 
that  there  are  here  and  there  cultures  of  LofHer  bacilli  in 
which  the  polar  granules  did  not  appear,  and  in  such  in- 
stances he  advises  the  examination  of  a  number  of  cover- 
slip  preparations.  He  found  also  isolated  bacilli  in  pseudo- 
diphtheria-bacillus  cultures  which  contained  the  polar 
granules  stained  by  Neisser's  method.  He  advises  in  all 
cases  of  doubt  that  cultures  be  made  to  determine  the 
production  of  acid  or  alkali  in  bouillon,  preferably  litmus 
bouillon,  and  inoculation  into  animals,  before  a  definite 
diagnosis  is  made. 


36  Comparative  Studies 

During  the  past  two  years  I  have  been  engaged  upon 
a  comparative  study  of  the  pseudo-diphtheria  bacillus,  the 
xerosis  bacillus,  and  the  Loffler  bacillus,  with  special  ref- 
erence to  the  differentiation  of  thesei  groups  of  organisms. 
My  observations  have  been  made  on  cultures  derived  from 
various  sources,  as  the  urine  in  health  and  disease,  the 
normal  conjunctiva,  the  nose  and  throat  in  catarrhal  in- 
flammations, the  skin  in  impetigo,  and  from  secretions  of 
the  vagina  in  metritis. 

In  January,  1896,  a  slight  polyuria  with  symptoms  of 
mild  cystitis,  accompanied  by  a  slight  rise  in  temperature 
in  the  evening,  led  me  to  undertake  the  bacteriological 
study  of  my  own  urine.  I  at  once  discovered  a  bacillus 
which,  in  its  morphological  characters,  closely  resembled 
the  diphtheria  bacillus.  Repeated  examinations  of  the 
urine  disclosed  the  same  organism  persisting  for  some  time. 
When  studied  on  the  different  culture  media  it  was  found 
to  grow  on  blood  serum  in  the  form  of  a  small,  round, 
whitish  colony,  with  no  tendency  to  coalesce  after  many 
days.  On  agar-agar  the  growth  was  in  the  form  of  very 
small,  pearly-white,  dry,  round  colonies  without  coales- 
cence. In  gelatin  stab  cultures  there  was  at  times  a  very 
slight  growth,  as  shown  by  the  whitish  line  along  the 
inoculation  stab.  Sometimes  no  growth  could  be  observed 
on  gelatin.  On  potato  only  a  few  bacilli  could  be  found 
on  scraping  the  surface  after  forty-eight  hours — and  ap- 
parently there  was  no  growth.  In  bouillon  there  was 
only  a  very  slight  growth,  as  only  a  few  bacilli  could  be 
found.  There  was  no  clouding  of  the  bouillon.  In 
litmus-milk  there  was  only  a  very  slight  growth.  At 
times  the  color  of  the  milk  was  changed  to  a  deeper  blue. 
In  rosolic  acid  solution  there  was  no  growth.  No  bacilli 
could  be  found.  The  color  of  the  solution  remained  the 
same.  In  peptone  solution  there  was  no  growth  ;  no 
bacilli  could  be  found.  There  was  no  indol  production.  In 
the  course  of  a  month  the  polyuria  and  fever  gradually 


Upon  the  Pseudo- Diphtheria,  etc.  37 

subsided,  and  no  further  attention  was  paid  to  the  matter. 
During  May,  1897,  there  was  a  return  of  the  symptoms, 
but  in  milder  form.  On  examination  the  same  organism 
was  again  found  in  the  urine.  It  was  found  repeatedly  for 
over  a  month.  Whether  the  organism  really  came  from 
the  bladder  or  not  it  is  impossible  to  state.  It  was  found 
in  cultures  made  from  the  meatus,  but,  judging  from  the 
symptoms,  I  am  inclined  to  believe  it  was  present  in  the 
bladder  itself.  Careful  study  of  the  organisms  isolated 
from  my  urine  in  May  revealed  no  biological  or  morpho- 
logical differences  from  the  organisms  isolated  in  January. 
Growth  always  took  place  most  readily  at  the  body  tem- 
perature ;  though  after  growth  had  once  become  established 
on  ordinary  media  it  was  found  possible  to  grow  the  organ- 
ism at  the  room  temperature.  Morphologically,  when 
grown  on  agar-agar  or  blood  serum  the  organism  appeared 
as  a  short,  slender  rod,  sometimes  showing  a  tendency  to 
clubbing  at  the  ends,  which  stained  more  deeply  at  the 
ends  than  elsewhere ;  usually,  when  stained  with  dilute 
methylene  blue,  the  bacilli  were  marked  with  alternate 
bands  of  deeply-stained  and  unstained  areas.  Usually,  the 
longer  and  more  club-shaped  organisms  showed  the  striae 
best.  It  was  never  seen  to  grow  visibly  on  potato.  In 
litmus-milk  there  was  sometimes  a  change  to  a  deeper  blue 
color.  There  was  not  not  always  a  growth  on  gelatin. 
The  color  of  the  rosolic  acid  solution  always  remained  un- 
changed. In  bouillon  there  was  at  times  a  very  slight 
flocculent  sediment  at  the  bottom  of  the  tubes.  On  blood 
serum  no  coalescence  of  the  small  round  colonies  was  ob- 
served at  any  time.  The  bacilli  appeared  to  stain  best 
with  aqueous  methylene  blue,  but  also  stained  well  with 
Loffler's  alkaline  methylene  blue.  They  stained  moder- 
ately well  with  aqueous  fuchsin  ;  and  more  deeply  with 
carbol-fuchsin.  They  stained  deeply  by  Gram's  method. 
By  staining  lightly  with  aqueous  methylene  blue  and 
counterstaining  with  Bismarck  brown,  small  bluish  gran- 


38  Comparative  Studies 

ules  were  noticeable  in  the  ends  of  the  club-shaped  bacilli, 
while  the  body  of  the  bacillus  had  a  faint  brownish  tint. 

DERIVATION  OF  GUI/TURKS  OF  THE  XEROSIS  BACILLUS 

AND  OF  THE  PSEUDO-DIPHTHERIA   BACILLUS. 

Cultures  i,  2  and  3  were  isolated  from  my  own  urine. 
Culture  4  was  isolated  from  the  urine  of  Mr.  A.,  a  healthy 
person.  Cultures  5,  6  and  8  were  isolated  from  the  con- 
junctival  sac  of  my  own  eyes.  Culture  9  was  isolated  from 
the  conjunctival  sac  of  Mr.  F.,  a  healthy  person;  culture 
10  from  the  conjunctival  sac  of  Mr.  H.,  aj  healthy  person. 
Culture  1 1  was  isolated  from  a  vaginal  discharge  taken 
from  the  case  of  Miss  C.,  in  the  University  Hospital. 
Culture  1 2  was  isolated  from  the  urine  of  a  patient  of  Dr. 
A.  Culture  13  was  isolated  from  the  urine  of  a  case  of 
catarrhal  jaundice.  These  cultures  were  obtained  during 
May  and  June,  1897.  Culture  14  was  isolated  from  the 
urine  of  Dr.  P.,  a  healthy  person.  Cultures  15  and  16 
were  isolated  from  the  conjunctival  sac  of  my  left  and  right 
eyes  respectively.  Culture  17  was  obtained  from  my  own 
urine.  Culture  18  was  derived  from  the  same  source,  and 
culture  19  from  my  nose,  which  appeared  to  be  in  a  healthy 
condition  at  the  time,  during  October  and  November,  1897. 
Culture  20  was^isolated  from  the  vaginal  secretions,  metritis, 
of  an  operative  case  in  the  Maternity  Department  of  the 
University  Hospital,  that  at  the  time  was  suffering  from 
septicaemia.  Streptococci  were  also  present  in  large  quan- 
tities. Culture  21  was  derived  from  a  small  abrasion  on 
my  knee,  which  at  the""time  showed  a  slight  tendency  to 
suppuration.  Staphylococci  were  also  present  at  the  time. 
Cultures  22  and  23  were  obtained  from  small  patches  of  a 
scaly  eruption  (impetigo  ?)  on  my  forearm  which  had  been 
noticed  for  several  months.  It  was  accompanied  with  but 
very  slight  itching  and  was  removed  after  several  days' 
treatment  'of  a  1-500  solution  of  bichloride  of  mercury, 


Upon  the  Pseudo- Diphtheria ,  etc.  39 

leaving  a  slightly  brownish,  glistening  spot  which  disap- 
peared after  several  days.  These  cultures  were  obtained 
during  November,  1897. 

Culture  24  was  obtained  December  28,  1897,  from  secre- 
tion of  my  nose,  during  an  attack  of  coryza  accompanied 
with  more  or  less  fever,  lassitude,  headache,  and  vague 
pains  throughout  the  system.  The  organism  appeared  to 
be  in  pure  culture,  and  was  found  plentifully  on  making 
cover-slip  preparations  from  the  secretions.  Culture  25 
was  derived  from  the  same  source,  a  few  days  later. 

Cultures  26  and  27  were  isolated  January  4,  1898,  from 
secretions  of  my  nose  during  an  attack  of  la  grippe. 
There  was  a  profuse  muco-purulent  discharge  from  the 
nostrils,  with  acute  pharyngitis  and  tonsilitis,  some  dis- 
phagia,  considerable  headache,  chilliness,  aching  of  the 
back  and  limbs,  with  considerable  fever.  The  prostration 
was  greater  than  in  the  attack  just  preceding  this.  Along 
with  these  organisms  were  found  streptococci  in  consider- 
able abundance.  The  nature  of  the  symptoms  and  the 
character  of  the  discharge  were  markedly  different  from 
those  of  the  previous  attack.  Whether  these  differences 
were  due  to  the  presence  of  the  streptococcus  in  addition 
to  the  pseudo-diphtheria  bacillus,  or  whether  they  were 
due  to  the  streptococcus  alone,  or  to  greater  virulence  of 
the  pseudo-diphtheria  bacillus,  it  is  impossible  to  say. 

Cultures  28  and  29  were  again  derived  from  the  eruption 
on  my  arm,  January  31,  1898,  and  culture  30  from  my 
nose,  February  14,  at  the  beginning  of  another  attack  of 
la  grippe.  The  influenza  bacillus  was  also  present  in 
large  numbers,  and  during  the  attack  the  xerosis  bacillus 
disappeared  from  the  secretions  of  the  nose.  Culture  31 
was  derived  from  the  urine  of  Mr.  H.,  a  case  of  mild  cys- 
titis, April  7,  1898  ;  staphylococci  were  also  present.  ' 

Morphologically,  these  different  organisms  resemble  each 
other  very  closely,  with  the  exception  of  culture  13,  which, 
when  first  isolated,  showed  very  large  club-shaped  bacilli 


40  Comparative  Studies 

in  blood-serum  cultures,  but  after  growing  on  artificial 
media  for  some  months  it  showed  only  as  short,  plump 
bacilli  with  rounded  ends,  the  centre  of  which  did  not 
take  on  stain,  while  the  poles  were  deeply  stained. 

All  the  other  cultures  occur  as  short  bacilli  with  pointed 
ends,  sometimes  having  only  one  clear,  unstained  space  in 
the  centre  and  the  poles  deeply  stained ;  again  at  times 
they  are  made  up  of  alternate  bands  of  stained  and  un- 
stained material.  In  blood-serum  cultures  and  in  alkali- 
peptone  media  a  considerable  number  of  club-shaped  bacilli 
were  to  be  seen,  especially  in  cultures  20,  26,  27  and  29  ; 
less  markedly  so  in  cultures  18,  21,  23,  24,  30  and  31  ; 
while  the  remaining  cultures  only  showed  club-shaped 
bacilli  occasionally.  A  peculiarity  of  these  cultures  which 
had  been  noted  by  other  investigators,  is  the  tendency  they 
have  of  two  or  three  lying  side  by  side  in  cover-slip  prep- 
arations. 

Biologically,  these  cultures  show  greater  differences.  A 
most  striking  difference  is  noted  in  the  growth  on  agar- 
agar.  Cultures  20,  26,  27  and  29  grow  as  a  thick,  moist, 
glistening,  white  or  yellowish-white  layer  on  the  surface 
of  the  agar.  Culture  13  grows  as  a  thick,  moist,  glisten- 
ing, yellowish  layer  on  agar-agar.  All  the  other  cultures 
grow  as  very \ninute,  dry,  pearly- white  colonies  and  show 
no  tendency  to  coalesce,  except  when  sown  very  thickly, 
when  they  grow  as  a  dull,  pearly-white  layer.  These 
differences  in  growth  were  somewhat  less  marked  on  blood- 
serum,  though  they  all  manifested  very  much  the  same 
characteristics  as  on  agar-agar. 

In  alkali-peptone  bouillon  (47)  the  same  cultures  show 
characteristics  which  correspond  with  the  characteristics 
on  agar-agar  and  on  blood-serum.  Cultures  20,  26,  27  and 
29  grow  with  a  thick  yellowish-white  mycoderm,  which 
sinks  to  the  bottom  of  the  tube  after  several  days,  while 
cultures  18  and  21  show  a  very  thin  mycoderm.  The 
remaining  cultures  show  a  very  fine  flocculent  deposit 


Upon  the  Pseudo- Diphtheria,  etc.  41 

along  the  sides  and  at  the  bottom  of  the  tube,  but  no 
mycoderm. 

When  grown  in  litmus-milk  considerable  variation  was 
noticed  with  regard  to  the  reaction  of  the  milk  after  grow- 
ing at  34°  C.  for  ten  days.  Under  the  same  conditions 
cultures  13,  14,  19,  23,  25,  26,  27  and  29  produced  an 
alkaline  reaction  which  was  noticeable  in  most  instances 
after  twenty-four  hours,  while  cultures  5,  15,  16,  18,  20,  21 
and  24  produced  an  acid  reaction  which  was  noticeable 
only  after  growing  for  four  days. 

When  grown  in  neutral  litmus  glucose  bouillon  more 
satisfactory  results  were  obtained  with  regard  to  the  pro- 
duction of  acids,  by  the  different  cultures,  than  with  the 
use  of  litmus  milk.  With  the  exception  of  cultures  23, 
26,  27  and  29,  all  produced  an  acid  reaction  in  this  medium, 
after  growing  in  the  incubator  for  ninety-six  hours.  Some 
of  the  cultures  showed  a  slight  acid  reaction  after  seventy- 
two  hours,  while  culture  20  showed  a  marked  acid  reaction 
after  twenty-four  hours'  growth.  The  extent  of  acid  pro- 
duction by  this  culture  was  apparently  equal  to  that  of 
two  cultures  of  the  I/5ffler  bacillus  grown  under  the  same 
conditions.  Cultures  21  and  31  produced  almost  the  same 
degree  of  acidity  as  the  cultures  of  the  Loffler  bacillus, 
though  not  as  rapidly  as  in  the  case  of  culture  20.  Cul- 
tures 23  and  29  failed  to  change  the  reaction  of  the  medium. 
Cultures  26  and  27  showed  a  distinct  alkaline  reaction 
after  growing  ninety-six  hours.  These  were  the  only  cul- 
tures which  produced  an  alkaline  reaction  in  this  medium. 

When  grown  on  potato,  cultures  5,  18,  20,  21,  23,  24,  26 
and  29  showed  a  slight  growth  after  ten  days  at  34°  C.  A 
similar  growth  was  noticed  in  cultures  of  the  Loffler 
bacillus  when  grown  under  the  same  conditions.  With 
the  exception  of  cultures  20  and  21  the  growth  was  barely 
perceptible.  It  is  probable  that  the  potato  was  somewhat 
alkaline,  as  the  Loffler  bacillus  does  not  grow  on  acid 
potato. 


42  Comparative  Studies 

The  colonies  on  agar  plates  of  cultures  13,  20,  26,  27 
and  29  were  somewhat  more  dense  than  those  of  the  Loffler 
bacillus,  and  they  were  also  slightly  yellowish  in  color ; 
otherwise  they  resembled  those  of  the  Loffler  bacillus. 
The  colonies  of  the  other  cultures  resembled  those  of  the 
L6ffler  bacillus  quite  closely. 

The  cultures  were  studied  for  some  time  with  regard  to 
the  effect  of  Neisser's  differential  stain.  They  were  grown 
on  Loffler's  blood  serum  at  a  temperature  of  34°  C.  for  from 
ten  to  twenty  hours,  and  were  then  examined.  Cultures 
5,  1 8,  20,  21,  23,  26  and  29  showed  more  than  the  usual 
number  of  bacilli  with  deeply-stained  polar  granules  ;  all 
the  other  cultures,  except  13,  showed  only  an  occasional 
bacillus  with  the  polar  granules  stained.  Culture  13 
seemed  to  show  the  granules  stained  as  uniformly  as  the 
cultures  of  the  Loffler  bacillus,  though  the  morphological 
characters  of  this  bacillus  are  different  from  those  of  the 
Loffler  bacillus.  These  results  coincide  with  the  state- 
ments of  Neisser  that  the  cultures  of  the  pseudo-diphtheria 
bacillus  show  only  an  occasional  bacillus  with  the  polar 
granules  stained.  Control  cultures  of  the  Loffler  bacillus 
grown  under  the  same  conditions  always  showed  most  of 
the  bacilli  with  the  polar  granules  stained,  or  even  with 
three,  four  or  more  granules  in  each  bacillus. 

Experiments  were  made  with  a  large  number  of  different 
aniline  stains,  in  watery  solutions,  with  and  without  de- 
colorizing agents,  with  the  hope  of  discovering  additional 
methods  of  differentiation  between  these  groups  of  organ- 
isms. No  very  satisfactory  results  were  obtained,  though 
the  following  stains  served  to  differentiate  between  the 
pseudo-diphtheria  bacilli  and  the  Loffler  bacilli ;  but  the 
differentiation  was  in  no  case  more  definite  than  by  Neis- 
ser's method  : 

i.  Loffler's  alkaline  methylene  blue,  one  minute,  wash 

in  water. 
Lugol's  solution,  one  minute,  wash  in  water. 


Upon  the  Pseudo- Diphtheria ,  etc.  43 

I/5ffler  bacilli — granules  blue,  bacilli  brown. 
Pseudo-diphtheria  bacilli — granules  blue  (if  any  are 
present),  bacilli  brown. 

2.  Koch-Bhrlich  gentian  violet,  one  minute,  wash  in 

water ;  10  per  cent  acetic  acid,  one  minute,  wash 

in  water. 

IxJffler  bacilli — granules  violet,  bacilli  decolorized. 
Pseudo-diphtheria  bacilli — granules  violet  (if  any  are 

present),  bacilli  decolorized. 

3.  Watery  solution  of  dahlia,  one  minute. 

lyoffler  bacilli — granules  deeply  stained,  bacilli  light 

violet. 
Pseudo-diphtheria    bacilli — bacilli     deeply    stained, 

clear  space  in  centre. 

4.  Watery  solution  of  methyl  violet,  one  minute. 
IvofHer   bacilli — granules   deep    violet,    bacilli    less 

deeply. 

Pseudo-diphtheria  bacilli — bacilli  deep  violet,  homo- 
geneous. 

5.  Watery  solution  of  crystal  violet,  one  minute. 
Same  results  as  with  methyl  violet. 

6.  Watery  solution  of  crystal  violet,  one  minute,  wash 

in  water ;  10  per  cent  solution  of  iodin  in  water, 
one  minute,  wash  in  water. 

Same  results  as  with  watery  solution  alone,  except 
that  the  pseudo-diphtheria  bacilli  show  the  seg- 
mentation. 

From  the  results  obtained  in  the  study  of  the  biological 
characters  of  the  different  cultures  it  is  evident  that  we 
are  dealing  with  two  distinct  groups  of  organisms  ;  cultures 
20,  26,  27,  29  and  31,  from  their  growth  on  agar,  blood 
serum  and  alkali-peptone  bouillon,  belong  to  the  group  of 
so-called  pseudo-diphtheria  bacilli,  while  all  the  other  cul- 
tures belong  to  the  group  of  xerosis  bacilli,  except  culture 
13,  which  I  have  not  classified  definitely,  though  it  probably 


44  Comparative  Studies 

also  belongs  to  the  former  group.  These  groups  of  organ- 
isms are  evidently  closely  related  to  the  I/5ffler  bacillus. 
I  am  inclined  to  believe,  with  Fraenkel,  Escherich,  Spronck 
and  Trumpp,  that  these  organisms  are  distinct  organisms, 
and  that  they  are  not  a  virulent  L,6ffler  bacilli.  There  is 
so  far  no  evidence  to  believe  that  they  are  attenuated 
Iv6ffler  bacilli.  All  the  experimental  evidence  points  to 
the  opposite  opinion.  Roux  and  Yersin,  L,6ffler  and  von 
Hofmann-Wellenhof  were  inclined  to  believe  they  were 
non-virulent  Loffler  bacilli ;  this  opinion  being  not  the  one 
now  generally  held  by  bacteriologists. 

TABLE  I. 

The  following  table  shows  the  results  obtained  with 
some  of  the  cultures  which  were  carefully  studied  in  their 
behavior  on  the  different  culture  media,  presenting  the 
comparative  results  in  a  more  graphic  form  : 


Upon  the  Pseudo- Diphtheria,  etc. 


45 


rce  of 
lture. 


8     8 


3   88 


5S 

12  TC. 


.s  g   3 

5D|S 

IB  1 

>     < 


+1+ 


I+I+ 


+I    1  +1+ 


•+ 


p      p      p      a      8  8 

^  -g  ^  -g  ^  *j  ^  -g  g  g* 


s  -  « 

.•3    ^nd 


i-T»4i-J 


+       1        1        1        I 


3s 


1       §     +      1 


+     +      1      +      1      +    II   1 


•S     ^    ^ 

•|   -|   .2  g 

•g   I 


o<u 
«  <u  S    ^ 

><        2 


M(NW 

o;<ij<u 
^^^J 

333 


c          — 

•S  §*8  S-^ 

^r2     ?     0     ^ 

o     *3  ^, 


o 


o      in  "-1  ."«     10  »o  »o  io«SS 

«       w^'oo;  <u  <u  w«(U<u 

^<Ug^j  ^  JS4  ^^^j^ 

-  -  - 


Ag 
lon 


rt   S  S  S 


aajw 

sr««lsi8f8 


jaqran^j 


IOCS         rO>OVOOO 


O       M       «o 

N          M          M 


<N  W  M    N    W 


46  Comparative  Studies 

Most  of  the  cultures  under  observation  were  cultivated 
in  alkali-peptone  bouillon  (45)  for  several  months,  each 
culture  being  transplanted  into  fresh  media  at  intervals  of 
forty-eight  hours,  with  the  hope  of  increasing  their  viru- 
lence. A  number  of  the  cultures  produced  a  slight  sub- 
cutaneous edema  at  the  point  of  inoculation,  when  first 
isolated,  but  no  greater  degree  of  virulence  could  be  se- 
cured by  the  cultivation  in  alkali-peptone  bouillon,  though 
with  most  of  the  cultures  a  constant  reduction  in  the  body 
weight  of  the  animal  was  noticed  during  the  first  week  fol- 
lowing the  inoculations.  The  animals  usually  regained 
their  weight  during  the  second  week.  Occasionally 
an  animal  succumbed  after  inoculation,  but  as  far  as 
could  be  determined  death  was  always  due  to  some  inter- 
current  affection,  usually  a  low-grade  pneumonia  due  to 
staphylococcus  infection.  When  inoculated  into  the  perito- 
neal cavity  with  large  doses  (4  to  5  c.  c.)  of  alkali-peptone, 
bouillon  cultures,  forty-eight  hours  old,  several  of  the  ani- 
mals died.  In  the  autopsies  on  these  animals  slight  sub- 
cutaneous edema  was  sometimes  found.  The  adrenals 
were  at  times  found  to  be  slightly  hyperaemic.  When  in- 
oculated intra-peritoneally,  there  was  at  times  considera- 
ble sanguinous  fluid  in  the  peritoneal  cavity,  which  con- 
tained masses  of  the  bacilli.  Numerous  leucocytes  were 
found  filled  with  the  bacilli.  The  organisms  were  recov- 
ered in  cultures  made  from  the  peritoneal  fluid,  while  cul- 
tures from  the  different  organs  remained  sterile.  There 
were  nearly  always  extensive  adhesions  of  loops  of  intes- 
tine to  the  abdominal  wall  or  other  organs  in  the  animals 
inoculated  intra-peritoneally.  Aside  from  foci  of  inflam- 
mation in  the  lungs  in  some  cases,  and  occasionally  con- 
gestion of  the  spleen,  the  internal  organs  were  found  nor- 
mal. No  glandular  enlargements  were  noticed. 

Attempts  were  also  made  to  increase  the  virulence  of  the 
organisms  by  passing  the  cultures  through  several  animals, 
but  no  definite  results  were  obtained. 


Upon  the  Pseudo- Diphtheria,  etc.  47 

An  attempt  was  made  to  render  a  number  of  animals 
immune  to  the  several  cultures  by  inoculating  them  at 
intervals  of  four  to  ten  days  with  cultures  of  the  same 
organism,  and  then  after  such  treatment  for  six  weeks  to 
two  months  they  were  inoculated  with  cultures  of  the 
Loffler  bacillus.  The  results  obtained  do  not  indicate  that 
any  degree  of  immunity  had  been  acquired  from  any  of 
the  cultures  employed.  When  fatal  doses  of  the  Loffler 
bacillus  were  inoculated  subsequently,  they  died  from  ex- 
perimental diphtheria. 

The  following  are  the  results  of  the  autopsies  on  the 
animals  which  died  in  consequence  of  inoculations  of  the 
different  cultures,  in  the  attempt  to  increase  the  virulence 
by  passing  them  through  successive  cultures  of  alkali-pep- 
tone bouillon,  along  with  the  results  of  sections  of  the 
organs  of  these  animals  : 

Autopsy  on  guinea  pig  No.  7. — Died  nine  and  a  half 
days  after  inoculation  with  i  c.  c.  of  culture  13.  Not 
greatly  emaciated  ;  inguinal  and  axillary  glands  appear 
slightly  enlarged.  Abdominal  walls  at  site  of  inoculation  are 
slightly  hyperaemic.  Liver  seems  somewhat  hyperaemic. 
Spleen  is  enlarged  and  dark.  Kidneys  are  slightly  con- 
gested. Adrenals  are  large,  pale  and  brittle.  The  lungs 
are  congested  and  show  large  areas  here  and  there  that  are 
completely  hepatized.  Cultures  made  from  the  hepatized 
lung  show  [the  micrococcus  tetragenous.  Cultures  made 
from  the  other  organs  remained  sterile.  On  section  the 
liver,  kidneys  and  spleen  showed  more  or  less  congestion, 
aside  from  which  nothing  abnormal  was  noted.  The  lung 
showed  extensive  infiltration  into  the  air  cells,  with  some 
proliferation  of  the  connective-tissue  cells,  and  marked  con- 
gestion. 

Guinea  pig  No.  55. — Died  twenty-two  hours  after  re- 
ceiving a  second  inoculation  of  culture  27  (4  c.  c.)  into  the 
peritoneal  cavity.  The  subcutaneous  lymphatics  were  some- 
what congested.  There  was  a  large  amount  of  sero-sangui- 


48  Comparative  Studies 

nous  fluid  in  the  peritoneal  cavity.  The  leucocytes  in 
this  fluid  contained  large  numbers  of  bacilli.  The  liver, 
kidneys,  lungs  and  adrenals  were  also  somewhat  congested. 
The  pericardial  sac  contained  a  considerable  amount  of 
clear  fluid.  The  bacilli  were  found  in  cultures  made  from 
the  peritoneal  cavity.  Cultures  from  all  the  organs  re- 
mained sterile.  On  section  the  lung  shows  here  and  there 
small  areas  of  infiltration.  The  adrenals  show  slight  areas 
of  cell  necrosis  and  portions  of  the  organ  seem  to  be 
completely  choked  with  blood,  showing  marked  conges- 
tion. Aside  from  the  congestion  nothing  abnormal  was 
noted  in  the  kidneys,  liver  or  spleen. 

Guinea  pig  No.  60. — Died  twenty-two  days  after  subcu- 
taneous inoculation  with  3  c.  c.  of  an  alkali-peptone  bouil- 
lon culture  of  25.  The  animal  is  emaciated  ;  the  subcu- 
taneous lymphatics  somewhat  hyperaemic ;  the  liver  dark 
and  enlarged  ;  the  gall-bladder  well  filled  with  pale  bile  ; 
the  kidneys  and  lungs  somewhat  congested.  The  spleen 
and  adrenals  appear  normal.  Cultures  made  from  the 
organs  remain  sterile.  On  section  the  lung  shows  consid- 
erable cellular  infiltration.  The  kidney,  liver  and  spleen 
are  congested  to  a  considerable  extent. 

Guinea  pig  No.  68. — Died  sixteen  hours  after  a  second 
inoculation  of  3  c.  c.  into  the  peritoneal  cavity  of  culture 
20.  Shows  subcutaneous  edema  quite  marked  ;  some  fluid 
in  the  pericardial  sac ;  adrenals  distinctly  hyperaemic ; 
kidneys  and  liver  congested.  The  retro-peritoneal  glands 
are  not  enlarged.  Cover-slips  prepared  from  the  peritoneal 
fluid  show  numerous  clumps  of  the  bacilli  and  many  leu- 
cocytes loaded  with  the  bacilli.  Cover-slips  from  the  liver, 
kidney  and  blood  were  negative.  Cultures  from  the  peri- 
toneal fluid,  liver  and  blood  showed  the  bacilli.  The  liver 
shows  here  and  there  rather  1  arge  areas  of  cell  necrosis.  In 
the  centre  of  these  necrotic  spots  there  is  complete  disinte- 
gration of  the  cellular  elements.  Along  the  margins  some 
of  the  cells  are  partially  disintegrated,  showing  a  pale 


Upon  the  Pseudo- Diphtheria,  etc.  49 

nucleus,  or  a  fragmented  nucleus  ;  others  show  fairly  well 
in  outline.  The  lung  shows  here  and  there  small  areas  of 
cellular  infiltration.  Aside  from  the  congestion,  nothing 
abnormal  was  noted  in  the  kidneys. 

Guinea  pig  No.  65. — Died  ten  days  after  being  inocu- 
lated with  4  milligrams  of  a  24-hour-old  blood-serum  cul- 
ture of  20.  The  liver  and  kidneys  showed  considerable 
congestion.  The  bladder  was  very  much  distended  with 
pale,  straw-colored  urine.  In  the  interior  of  the  bladder 
was  what  appeared  to  be  an  old  pseudo-membranous  cast 
of  the  lining  membrane.  It  was  whitish  in  color,  soft, 
friable,  and  stained  imperfectly,  the  stained  matter  looking 
like  cell  debris.  A  few  bacilli  were  found  in  this  matter. 
The  mucous  membrane  of  the  bladder  was  hyperaemic. 
Numerous  bacilli  were  found  in  cover-slip  preparations 
made  from  the  seat  of  inoculation.  Cultures  made  from 
the  internal  wall  of  the  bladder  and  from  the  pseudo-mem- 
brane in  the  bladder  showed  the  pseudo-diphtheria  bacillus. 

Guinea  pig  No.  76. — Had  been  inoculated  at  intervals 
for  about  two  months,  five  in  number,  with  alkali-peptone 
bouillon  cultures  of  26,  with  the  idea  of  rendering  it  im- 
mune. The  animal  died  about  thirty  hours  after  the  last 
inoculation  into  the  peritoneal  cavity.  On  autopsy  a  small 
hemorrhagic  point  was  observed  in  the  abdominal  wall,  at 
the  point  of  puncture  with  the  needle,  in  which  the  bacilli 
were  found.  There  was  a  large  amount  of  sanguinous 
fluid  in  the  peritoneal  cavity.  Adhesions  of  the  abdomi- 
nal organs  and  recent  peritonitis  were  also  noted.  The 
organs  were  congested  ;  the  adrenals  were  slightly  hyper- 
aemic. The  pericardial  sac  also  contained  a  little  clear 
fluid.  Cover-slips  made  from  the  peritoneal  fluid  showed 
leucocytes  filled  with  bacilli.  Cultures  from  the  exudate  in 
the  peritoneal  cavity  showed  the  pseudo-diphtheria  bacillus. 

Guinea  pig  No.  98. — Was  inoculated  subcutaneously 
with  4  c.  c.  of  a  48-hour-old  alkali-peptone  bouillon  culture 
of  27,  and  died  eighteen  days  later.  On  autopsy  no  lesion 


50  Comparative  Studies 

was  found  at  the  site  of  inoculation.  There  was  no  fluid 
in  the  internal  cavities.  Spleen,  kidneys  and  liver  were 
normal  ;  adrenals  slightly  hyperaemic.  The  lungs  showed 
areas  of  inflammation.  Cover-slips  made  from  the  lungs 
showed  micrococci.  On  section,  the  liver,  kidneys,  spleen 
and  adrenals  were  found  normal.  The  lung  was  found  to 
be  very  much  conjested,  with  infiltration  of  cellular  ele- 
ments, showing  areas  of  complete  consolidation. 

It  will  be  observed  that  the  results  obtained  in  the  autop- 
sies on  these  animals  are  somewhat  variable.  On  the  whole, 
the  lesion's  observed  were  dissimilar  from  those  found  in  ex- 
perimental diphtheria.  In  several  of  the  animals  some  slight 
subcutaneous  edema  was  noted,  but  nothing  very  marked. 
There  was  in  no  instance  distinct  glandular  enlargement, 
either  of  the  external  lymphatic  glands  or  of  the  retro- 
peritoneal  glands,  as  noted  usually  in  inoculations  with  the 
Loffler  bacillus.  The  appearance  of  the  adrenals,  while  at 
times  somewhat  hyperaemic,  was  usually  the  contrary — 
that  is,  they  were  rather  pale  and  anaemic. 

Sections  of  the  organs  of  these  animals  presented  no  dis- 
tinctive features.  Usually,  death  supervened  after  two  or 
more  inoculations  with  the  same  organism,  at  intervals  of 
five  to  seven  days.  The  most  common  feature  in  these 
autopsies  was  the  apparent  lowered  vitality  of  the  animal, 
as  the  result  of  the  inoculations  ;  and  in  several  instances 
the  animal  evidently  succumbed  to  secondary  inflamma- 
tion, more  particularly  inflammation  of  the  lung,  as  the 
result  of  the  invasion  of  pathogenic  cocci. 

The  results  of  the  inoculation  experiments  afford  no  as- 
sistance in  classifying  these  organisms.  Neither  do  the 
results  of  the  growth  in  the  different  culture  media  present 
features  which  are  uniformly  constant  with  regard  to  the 
different  cultures.  However,  from  their  biological  charac- 
ters, as  noted  more  particularly  on  agar-agar  and  blood 
serum  with  regard  to  the  macroscopic  appearance  of  the 
cultures,  we  may  classify  them  into  two  groups,  more  or  less 


Upon  the  Pseudo- Diphtheria,  etc.  51 

distinct,  namely  :  those  which  grow  as  a  distinct  layer  upon 
these  media,  forming  a  rather  thick,  creamy-white  surface- 
growth,  and  the  group  comprising  those  which  grow  only, 
or  usually  at  least,  as  small,  pearly-white,  isolated  colonies 
on  these  media.  The  former  group  comprises  the  organ- 
ism which  is  usually  designated  as  the  pseudo-diphtheria 
bacillus,  or  Hofmann  bacillus.  The  latter  group  comprises 
what  is  usually  known  as  the  xerosis  bacillus.  Morpholog- 
ically, these  two  groups  of  organisms  resemble  each  other 
very  closely,  and  they  also  resemble  the  L,6ffler  bacillus 
quite  closely, — so  much  so  that  their  occurrence  in  cul- 
tures made  for  the  diagnosis  of  diphtheria  is  sure  to  lead 
to  errors  in  diagnosis  unless  the  precaution  is  taken  to 
apply  Neisser's  differential  stain.  Our  experience  has  been, 
however,  that  where  this  differential  staining  method  has 
been  systematically  employed  a  safe  differential  diagnosis 
can  be  made,  from  the  fact  that  neither  of  these  groups 
of  organisms  shows  the  polar  granules  of  Neisser  when 
grown  under  the  prescribed  conditions. 

There  has  been  for  some  time  considerable  controversy 
between  bacteriologists  with  regard  to  the  identity  of  these 
micro-organisms.  Loffler,  v.  Hofmann,  Roux  and  Yersin, 
Koch,  Dunbar  and  Abbott  are  of  the  opinion  that  these 
two  groups  of  organisms  are  merely  modified  forms  of  the 
Loffler  bacillus.  On  the  other  hand,  Hueppe,  Fraenkel, 
Escherich,  Spronck  and  Trurnpp,  among  others,  belonging 
to  what  is  called  the  separatist  school,  hold  that  these  two 
groups  of  organisms  are  entirely  different  from  the  L,6ffler 
bacillus,  and  that  diphtheria  is  always  produced  by  viru- 
lent diphtheria  bacilli,  and  that  these  two  groups  of  organ- 
isms never  increase  in  virulence  to  such  an  extent  as  to  pro- 
duce diphtheria.  Most  of  the  later  investigators  are  ready 
to  accept  the  teaching  of  the  separatist  school,  by  the  light 
of  the  more  definite  methods  now  being  employed. 

From  the  results  of  my  studies  of  these  organisms  I  have 
reached  the  conclusion  that  we  have  to  deal  with  a  large 


52  Comparative  Studies 

group  of  micro-organisms,  at  the  head  of  which  is  the 
virulent  L<ofiler  bacillus,  which  may  occur  with  several 
distinct  variations,  as  shown  in  cultures  derived  from  dif- 
ferent cases  of  diphtheria.  The  most  marked  differences 
with  regard  to  the  L,6ffler  bacillus  are,  first,  long,  slender 
bacilli  which  show  a  tendency  to  the  formation  of  rather 
large,  club-shaped  organisms  on  blood-serum  ;  and  second, 
rather  short,  thick,  ovoid  bacilli  which  show  the  club- 
shaped  forms  only  occasionally. 

At  the  other  extreme  of  this  large  group  of  micro-organ- 
isms is  the  xerosis  bacillus.  Between  these  two  extremes, 
the  types  of  which  are  the  L,6ffler  bacillus  on  the  one 
hand  and  the  xerosis  bacillus  on  the  other,  we  have 
many  variations  in  type,  as  shown  by  the  modifications 
of  biological  and  morphological  characters.  The  organisms 
which  I  have  been  studying,  belonging  more  distinctly 
to  the  group  of  pseudo-diphtheria,  or  Hoffmann  bacilli,  are 
cultures  Nos.  13,  20,  26,  27,  29  and  31.  All  the  other  cul- 
tures studied  belong  to  the  group  of  xerosis  bacilli. 

I  believe  it  would  be  preferable  to  designate  as  Hofmann 
bacilli  all  those  so-called  pseudo- diphtheria  bacilli  which 
grow  as  a  thick,  creamy-white  layer  on  agar-agar  and  on 
blood  serum.  It  is  evident  that  these  organisms  are  not 
a-virulent  LofHer  bacilli,  and  therefore  have  no  direct  con- 
nection with  the  disease  processes  in  cases  of  diphtheria. 
If  they  are  capable  of  producing  any  lesions  whatever 
these  are  of  a  mild  character  and  largely  local  in  their 
manifestation.  The  conditions  existing  in  my  nose  and 
throat  at  the  time  of  isolating  several  of  the  cultures  stud- 
ied were  of  the  nature  of  a  mild  catarrhal  inflammation. 

In  this  connection  I  wish  to  thank  Dr.  A.  C.  Abbott, 
Director  of  the  Laboratory,  for' valuable  suggestions  and 
advice  given  during  the  investigation. 


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(School  of  Arts);  No.  2  (Towne  Scientific  School);  No.  3  (Courses 
for  Teachers) . 

Report  of  the  Provost  (published  in  January). 

Group  II. — Serial  Publications. 
Series  in  Philology,  Literature  and  Archaeology. 
Series  in  Philosophy. 

Series  in  Political  Economy  and  Public  Law. 
Series  in  Botany. 
Series  in  Zoology. 
Series  in  Mathematics. 
University  Bulletin  (monthly). 

Group  III. — Occasional  Publications. 

Reports  of  the  fluseums  of  Archaeology  and  Paleontology. 
Theses  presented  for  the  Degree  of  Doctor  of  Philosophy. 

•\Group  IV.— Affiliated  Publications. 

Annals  of  the  American  Academy  of  Political  and  Social  Science. 
Americana  Qermanica  (quarterly). 
Bulletin  of  the  Free  Museum  of  Science  and  Art. 
Translations  and  Reprints  from  the  Original  Sources  of  European 
History. 

EXPLANATORY. 

Group  I  consists  of  publications  issued  annually  under  the  direct 
auspices  of  the  Provost  and  Trustees. 

The  University  Catalogue  is  a  volume  of  about  500  pp.     It  contains 
detailed  information  concerning  all  departments,  lists  of  officers  and 

*  Beginning  with  New  Series,  No.  i. 
t  For  exchange  purposes  only. 

(55) 


56          Publications  of  the  University  of  Pennsylvania. 

students,  with  addresses,  etc.  No  charge  is  made  for  the  Catalogue,  but 
in  all  cases  requests  for  a  copy  by  mail  must  be  accompanied  by  ten  cents 
in  stamps  to  cover  postage. 

The  Fasciculus  of  each  department  contains  information  concerning 
that  department  only;  while  the  three  College  Circulars  of  Information, 
covering  respectively  the  School  of  Arts,  the  Towne  Scientific  School, 
and  the  Courses  for  Teachers,  are  in  like  manner  restricted  as  to  their 
contents.  The  Fasciculi  and  College  Circulars  are  published  separately 
after  the  University  Catalogue,  of  which  they  are,  to  a  large  extent, 
reprints.  Single  copies  are  mailed  free  upon  request. 

The  Report  of  the  Provost,  made  by  him  annually  to  the  Corporation, 
constitutes  a  general  review  of  University  activities  during  the  year,  and 
contains  inter  alia  reports  from  the  Treasurer  and  the  several  Deans. 
Single  copies  are  mailed  free  upon  request. 

Group  II  consists  of  a  number  of  serial  publications  in  the  several 
fields  of  literature,  science  and  philology.  They  are  issued  in  separate 
series  at  irregular  intervals  (for  the  most  part),  and  represent  the  results 
of  original  research  by,  or  under  the  direction  of,  members  of  the  staff 
of  instruction  of  the  University.  A  complete  list  of  these  publications 
to  date,  with  prices  attached,  is  printed  at  length  following.  They  are 
published  under  the  editorial  supervision  of  the  University  Publications 
Committee. 

Group  HI  consists  of  occasional  publications,  such  as  reports  of  the 
various  University  departments  (where  printed  separately),  and  certain 
theses  presented  in  partial  fulfillment  of  the  requirements  for  the  degree 
Doctor  of  Philosophy. 

Group  IV  consists  of  affiliated  publications,  issued  as  separate  peri- 
odicals, not  under  the  control  of  the  University,  but  edited  in  part  by 
officers  of  the  University  of  Pennsylvania.  Copies  are  obtainable  from 
the  University  only  through  the  medium  of  exchange  (see  Exchange 
Bureau,  below). 

EXCHANGE  BUREAU. 

The  University  of  Pennsylvania  desires  to  extend  its  system  of  exchang- 
ing publications  with  other  similar  institutions  and  learned  societies, 
both  at  home  and  abroad. 

For  convenience  in  correspondence,  the  following  statement  is  made: 

To  those  educational  institutions  and  learned  societies  which  issue  only 
annual  catalogues,  reports,  or  similar  publications,  the  University  of 
Pennsylvania  offers  in  exchange  all  those  of  its  own  publications  classed 
under  Group  1  and  III,  or  as  many  of  them  as  may  be  specified. 

To  those  educational  institutions  and  learned  societies  publishing  also 
results  of  original  investigations,  the  University  of  Pennsylvania  offers 
in  exchange  any  one  of  its  equivalent  series  in  Groups  II  and  IV,  or  as 
many  of  them  as  may  be  mutually  agreed  upon  in  order  to  maintain  a 
proportionate  ratio  of  exchange. 


Publications  of  the   University  of  Pennsylvania.  57 

In  establishing  a  system  of  exchanges  with  any  other  institution,  the 
University  of  Pennsylvania  binds  itself  to  the  following  regulations: 

All  publications  agreed  upon  to  be  forwarded  from  Philadelphia  to 
address  furnished,  immediately  upon  issue,  free  of  expense  to  our  corre- 
spondent. 

In  return  the  University  requests  compliance  with  the  following: 

All  publications  to  be  forwarded  to  ' '  Library  of  the  University  of 
Pennsylvania,  Philadelphia,  Pa.,"  marked  "  Exchange  Bureau  "  in  lower 
left-hand  corner,  immediately  upon  issue,  free  of  expense  to  us. 


Orders  for  single  numbers,  or  sets  of  Serial  Publications  under  Group  II, 
and  all  correspondence  relating  to  the  publications  of  this  University, 
should  be  addressed  to 

J.  HARTLEY  MERRICK,  Assistant  Secretary, 

Station  B,  Philadelphia,  Pa. 


Philology,  Literature,  and  Archaeology 


Volume  I. 

1.  Poetic  and  Verse  Criticism  of  the  Reign  of  Elizabeth.     By  FELIX 

E.  SCHELLING,  Professor  of  English  Literature.     $1.00. 

2.  A  Fragment  of  the  Babylonian  "Dibarra"  Epic.      By  MORRIS 

JASTROW,  JR.,  Professor  of  Arabic.     60  cents. 

3.  a.  n/>dc  with  the  Accusative,    b.  Note  on  a  Passage  in  the  An- 

tigone.    By  WILLIAM  A.  LAMBERTON,  Professor  of  the  Greek  Language 
and  Literature.     50  cents. 

4.  The  Gambling  Games  of  the  Chinese  in  America  :   Fan  fan  and 

Pak   kop   piu.      By  STEWART   CULIN,    Secretary  of   the    Museum   of 
Archaeology  and  Paleontology.     40  cents. 

Volume  II. 

1.  Recent  Archaeological  Explorations  in  the  Valley  of  the  Dela- 

ware  River.       By   CHARLES   C.    ABBOTT,   Sometime  Curator  of   the 
Museum  of  American  Archaeology.     75  cents. 

2.  The  Terrace  at  Persepolis.     By  MORTON  W.  EASTON,  Professor  of 

English  and  Comparative  Philology.     25  cents. 

3.  The  Life  and  Writings  of  George  Gascoigne.    By  FELIX  E.  SCHEL- 

LING, Professor  of  English  Literature.     $l.oo. 

Volume  III. 

1.  Assyriaca.  By  HERMANN  V.  HILPRECHT,  Professor  of  Assyrian  and  Com- 

parative Semitic  Philology  and  Curator  of  Babylonian  Antiquities.     $1.50. 

2.  A  Primer  of  Mayan  Hieroglyphics.     By  DANIEL  G.  BRINTON,  Pro- 

fessor of  American  Archaeology  and  Linguistics.     $1.20. 


58  Publications  of  the   University  of  Pennsylvania. 

Volume  IV. 

1.  The  Rhymes  of   dower's  Confessio  Amanti.      By  MORTON  W. 

EASTON,  Professor  of  English  and  Comparative  Philology.     60  cents. 

2.  Social  Changes  in  the  Sixteenth  Century  as  Reflected  in  Con- 

temporary English  Literature.     By  EDWARD  P.  CHEYNEY,  Assist- 
ant Professor  of  History.     #1.00. 

3.  The  War  of  the  Theatres.     By  JOSIAH  H.  PENNIMAN,  Instructor  in 

English.     1 1.  oo. 

Volume  V.    $2.00. 

Two  Plays  of  Miguel  Sanchez  (surnamed  "El  Divino").    By 
HUGO  A.  RENNERT,  Professor  of  Romance  Languages  and  Literatures. 

Volume  VI.    $2.00. 

a.    The  Antiquity  of  Man  in  the  Delaware  Valley. 
•'.    Exploration  of  an  Indian  Ossuary  on  the  Choptank  River,  Dor- 
chester  Co.,  Md.       With  a  description  of  the  crania  discovered  by 
E.  D.  Cope;  and  an  examination  of  traces  of  disease  in  the  bones,  by 
Dr.  R.  H.  Harte. 

.  Exploration  of  Aboriginal  Shell  Heaps  on  York  River,  Maine. 
By  HENRY  C.  MERCER,  Curator  of  the  Museum  of  American  Arch- 
aeology. 


Philosophy 


i.  *  Sameness  and  Identity.     By  GEORGE  STUART  FULLERTON. 

a.  *On  the  Perception  of  Small  Differences.  With  special  reference  to 
the  Extent,  Force,  and  Time  of  Movement.  By  GEORGE  STUART  FULLER- 
TON  and  JAMES  McKEEN  CATTELL. 


Political  Economy  and  Public  Law 


t  Volume  I. 

1.  The  Wharton  School  Annals  of  Political  Science.      March,  1885. 

2.  The  Anti-Rent  Agitation  in  the  State  of  New  York.    1839-1846. 

By  EDWARD  P.  CHEYNEY. 

3.  Ground  Rents  in  Philadelphia.     By  EDWARD  P.  ALLINSON  and  B. 

PENROSE. 

4.  The  Consumption  of  Wealth.     By  SIMON  N.  PATTEN. 

*  Out  of  print. 

f  No  copies  available  for  exchange. 


Publications  of  the   University  of  Pennsylvania.  59 

5.  Prison  Statistics  of  the  United  States  for  1888.    By  ROLAND  P. 

FALKNER. 

6.  The  Principles  of   Rational  Taxation.  (Read  at  a  meeting  of  the 

Association,  November  21,  1889.)     By  SIMON  N.  PATTEN. 

7.  The  Federal  Constitution  of  Germany.  With  an  historical  introduc- 

tion, translated  by  EDMUND  J.  JAMES. 

8.  The  Federal  Constitution  of  Switzerland.    Translated  by  EDMUND 

J.  JAMES. 

*  Volume  II. 

9.  Our  Sheep  and  the  Tariff.     By  WILLIAM  DRAPER  LEWIS. 

*  Volume  III. 

IO.     The   German  Bundesrath.      A  Study  in  Comparative  Constitutional 

Law.    By  JAMES  HARVEY  ROBINSON. 
n.    The  Theory  of  Dynamic  Economics.     By  SIMON  N.  PATTEN. 

*  Volume  IV. 

12.  The  Referendum  in  America.    A  Discussion  of   Law-Making  by 

Popular  Vote.     By  ELLIS  PAXSON  OBERHOLTZER. 

Volume  V. 

13.  Currency  Reform.     By  JOSEPH  FRENCH  JOHNSON.    25  cents. 


CONTRIBUTIONS 

The  Botanical  Laboratory 


Volume  I— No.  i.    $2.00. 

(Plates  I-XIII.) 

1.  A  Monstrous  Specimen  of  Rudbeckia  hirta,  L.      By  J.  T.  ROTH- 

ROCK,  B.S.,  M.D. 

2.  Contributions  to  the  History  of  Dionaea  fluscipula,  Ellis.     By 

J.  M.  MACFARLANE,  D.Sc. 

3.  An  Abnormal  Development  of  the  Inflorescence  of  Dionaea.    By 

JOHN  W.  HARSHBERGER,  A.B.,  B.S. 

4.  Mangrove  Tannin.     By  H.  TRIMBLE,  Pn.M. 

5.  Observations  on  Epigaea  repens,  L.     By  W.  P.  WILSON,  D.Sc. 

6.  A  Nascent  Variety  of  Brunella  vulgaris,  L.     By  J.  T.  ROTHROCK, 

B.S.,  M  D. 

7.  Preliminary  Observations  on  the  Movements  of  the  Leaves  of 

Melilotus  alba,  L.,  and  other  plants.     By  W.  P.  WILSON,  D.Sc., 
and  J.  M.  GREENMAN. 

*  No  copies  available  for  exchange. 


60          Publications  of  the    University  of  Pennsylvania. 

Volume  I— No.  2.    $2.00. 

(Plates  XIV-XVII.) 

8.  Maize  :  A  Botanical  and  Economic  Study.     By  JOHN  W.  HARSH 

BERGER,  PH.D. 

Volume  I — No.  3.    $2.00. 

(Plates  XVIII-XXXVI.) 

9.  A  Chemico-Physiological  Study  of  Spirogyra  nitida.     By 

MARY  E.  PENNINGTON,  PH.D. 

10.  On  the  Structure  and  Pollination  of  the  Flowers  of  Eupato- 

rium  ageratoides  and  I£.   coelestinum.     By  LAURA    B. 
CROSS,  PH.D. 

1 1 .  Contributions  to  the  Life-History  of  Amphicarpaea  monoica. 

By  ADELINE  F.  SCHIVELY,  PH.  D. 


CONTRIBUTIONS    RROM 

The  Zoological  Laboratory. 

Volume  I— No.  r.     $2.00. 

The  Correlations  of  the  Volumes  and  Surfaces  of  Organisms. 

By  JOHN  A.  RYDER,  PH.D.     (Plate  I.) 
The  Growth  of  Euglena  Viridis  when  Constrained  Principally 

to    Two  Dimensions  of  Space.     By    JOHN    A.    RYDER,  PH.D. 

(Plate  II.) 
Descriptions  of  Three  New  Polychaeta  from  the  New  Jersey 

Coast.     By  J.  PERCY  MOORE.     (Plates  IIl-IV.) 

Volume  I— No.  2.    $2.00. 

On  the  Embryos   of  Bats.     By  HARRISON  ALLEN,  M.  D.     (Plates 

V-VIII.) 


Mathematics. 


Volume  I — JVo.  i.    40  cents. 

Contributions  to  the  Geometry  of  the  Triangle.  By  R.  J. 
ALEY,  A.  M. 

Properties  of  the  Locus  r  =  Constant,  in  space  of  n  Dimen- 
sions. By  PAUL  R.  HEYL,  B.  S. 


STAMPED  BELOW 


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